Materials for hydrophilic interaction chromatography and processes for preparation and use thereof for analysis of glycoproteins and glycopeptides

ABSTRACT

The invention relates to poly-amide bonded hydrophilic interaction chromatography (HILIC) stationary phases and novel HILIC methods for use in the characterization of large biological molecules modified with polar groups, known to those skilled in the art as glycans. The invention particularly provides novel, poly-amide bonded materials designed for efficient separation of large biomolecules, e.g. materials having a large percentage of larger pores (i.e. wide pores). Furthermore, the invention advantageously provides novel HILIC methods that can be used in combination with the stationary phase materials described herein to effectively separate protein and peptide glycoforms by eliminating previously unsolved problems, such as on-column aggregation of protein samples, low sensitivity of chromatographic detection of the glycan moieties, and low resolution of peaks due to restricted pore diffusion and long intra/inter-particle diffusion distances.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalPatent Application No. 61/920,677, filed Dec. 24, 2013, the disclosureof which is expressly incorporated herein by reference thereto.

BACKGROUND OF THE INVENTION

Hydrophilic interaction chromatography (or hydrophilic interactionliquid chromatography, HILIC) is a variant of normal phase liquidchromatography that partly overlaps with other chromatographicapplications such as ion chromatography and reversed phase liquidchromatography. The stationary phase of HILIC is a polar and hydrophilicphase which results in enhanced retention for polar analytes. The mobilephase of HILIC is a reversed-phase type high organic eluent, forexample, a mixture of water and acetonitrile. A mechanism of separatinganalytes in HILIC can be a combination of partitioning, ion exchange andreverse-phase chromatography.

Prior to the present invention, HILIC has seen limited use for theseparation and analysis of large proteinaceuous biomolecules, such asglycoproteins. The authors of T. Tetaz et al., J. Chomatogr., A 1218,5892-5896 (2011), explored the analysis of intact soluble proteins usingHILIC with mediocre results, presenting chromatograms indicative ofrelatively low resolution separations. Similarly, the authors of,“Separations of Intact Glycoproteins by HILIC” at the 33rd InternationalSymposium and Exhibit on the Separation and Characterization ofBiologically Important Molecules, Jul. 17-19, 2013 in Boston, Mass.,USA, expressed that glycoprotein separations by HILIC would be ofsignificant interest though at present this poses a significantchallenge. Their presented work was limited to separation of small (<20kDa) glycoproteins.

Moreover, Guillarme (“What You Need to Know About HILIC” Jul. 1, 2013LCGC NORTH AMERICA Volume 31, Issue 7, pp. 560-563) notes that the peakshape of large proteins (greater than 20 kDa) using HILIC may beunacceptable as, prior to the present invention, optimized wide-poreHILIC phases are not available.

There remains a need for HILIC materials designed for highefficiency/high resolution separations of large biomolecules (e.g.larger average pore diameters) along with improved chromatographicmethodologies for the analysis of such samples, including thosecontaining proteinaceous biomolecules modified with polar groups knownto those skilled in the art as glycans.

SUMMARY OF THE INVENTION

The invention provides poly-amide bonded HILIC stationary phases alongwith novel HILIC-related methods for use in the characterization oflarge biological molecules modified with polar groups, known to thoseskilled in the art as glycans. The novel stationary phase materials areuseful in chromatographic processes, e.g., hydrophilic interactionchromatography, and provide a number of advantages. Such advantagesinclude high resolution of large biomolecules, desirable retentivity andselectivity of glycans/glycoforms, and non-retention of interferinganalytes. The invention advantageously provides novel, poly-amide bondedmaterials designed for efficient separation of large biomolecules, e.g.materials having a large percentage of larger pores (i.e. wide pores).Most importantly, the invention advantageously provides novel materialsthat can be used in combination with the methods described herein toeffectively separate protein and peptide glycoforms by HILIC.

In one aspect, a porous material comprising a copolymer comprising atleast one hydrophilic monomer and a poly-amide bonded phase, wherein theaverage pore diameter is greater than or equal to about 200 Å, greaterthan or equal to about 250 Å, greater than or equal to about 300 Å, orgreater than or equal to about 450 Å.

In certain aspect, the porous material comprises a porous particle thatcomprises said copolymer. In another certain aspect, the porous materialcomprises a porous monolith that comprises said copolymer.

In another aspect, The hydrophilic monomer is3-methacryloxypropyltrichlorosilane,3-methacryloxypropylmethyldichlorosilane,3-methacryloxypropyldimethylchlorosilane,3-methacryloxypropyltrimethoxysilane,3-methacryloxypropylmethyldimethoxysilane,3-methacryloxypropyldimethylmethoxysilane,3-methacryloxypropyltriethoxysilane,3-methacryloxypropylmethyldiethoxysilane,3-methacryloxypropyldimethylethoxysilane,3-acryloxypropyltrichlorosilane, 3-acryloxypropylmethyldichlorosilane,3-acryloxypropyldimethylchlorosilane 3-acryloxypropyltrimethoxysilane,3-acryloxypropylmethyldimethoxysilane,3-acryloxypropyldimethylmethoxysilane, 3-acryloxypropyltriethoxysilane,3-acryloxypropylmethyldiethoxysilane,3-acryloxypropyldimethylethoxysilane, styrylethyltrichlorosilane,styrylethylmethyldichlorosilane, styrylethyldimethylchlorosilane,styrylethyltrimethoxysilane, styrylethylmethyldimethoxysilane,styrylethyldimethylmethoxysilane, styrylethyltriethoxysilane,styrylethylmethyldiethoxysilane, styrylethyldimethylethoxysilane,vinyltriethoxysilane, vinyltrimethoxysilane,N-(3-acryloxy-2-hydroxypropyl)-3-aminopropyl triethoxysilane,(3-acryloxypropyl) trimethoxysilane,O-(methacryloxyethyl)-N-(triethoxysilylpropyl)urethane,N-(3-methacryloxy-2-hydroxypropyl)-3-aminopropyl triethoxysilane,methacryloxy methyltriethoxysilane, methacryloxymethyl trimethoxysilane,methacryloxypropy methyldiethoxysilane, methacryloxypropylmethyldimethoxysilane, methacryl oxypropyltris (methoxyethoxy)silane,3-(N-styrylmethyl-2-aminoethylamino) propyltrimethoxysilanehydrochloride,

In yet another aspect, the poly-amide bonded phase is derived fromacrylamide, divinylbenzene, styrene, ethylene glycol dimethacrylate,1-vinyl-2-pyrrolidinone and tert-butylmethacrylate, acrylamide,methacrylamide, N,N′-(1,2-dihydroxyethylene)bisacrylamide,N,N′-ethylenebisacrylamide, N,N′-methylenebisacrylamide, butyl acrylate,ethyl acrylate, methyl acrylate, 2-(acryloxy)-2-hydroxypropylmethacrylate, N,N-bis(2-cyanoethyl)acrylamide,N-acryloyltris(hydroxymethyl)aminomethane, 3-(acryloxy)-2-hydroxypropylmethacrylate, trimethylolpropane triacrylate, trimethylolpropaneethoxylate triacrylate, tris[(2-acryloyloxy)ethyl] isocyanurate,acrylonitrile, methacrylonitrile, itaconic acid, methacrylic acid,trimethylsilylmethacrylate, N-[tris(hydroxymethyl)methyl]acrylamide,(3-acrylamidopropyl)trimethylammonium chloride,[3-(methacryloylamino)propyl]dimethyl(3-sulfopropyl)ammonium hydroxideinner salt,

In certain aspect of the invention, the porous material comprises asecond poly-amide bonded phase. In particular aspect, the secondpoly-amide bonded phase is derived from N,N-methylenebisacrylamide,N,N-ethylenebisacrylamide, N,N-propylenebisacrylamide,N,N-butylenebisacrylamide, N,N′-(1,2-dihydroxyethylene)bisacrylamide, or1,4-bis(acryloyl)piperazine.

In other aspect of the invention, the first poly-amide bonded phase ispresent in about 35 to about 99 mole % of the total poly-amide bondedphases and the second poly-amide bonded phase is present in about 65 toabout 1 mole % of the total poly-amide bonded phases.

In certain aspect, the porous material has a median pore diameter ofabout 100 Å to about 1000 Å, of about 300 Å to about 800 Å, or of about300 Å to about 550 Å. In particular aspect, the porous material has amedian pore diameter of about 300 Å.

In another certain aspect, the nitrogen content of the porous materialis from about 0.5% N to about 20% N, from about 1% N to about 10% N,from about 2% N to about 10% N, or from about 4% N to about 10% N.

According to one embodiment, the present invention provides a method forremoving or isolating a component from a mixture comprising: contactingthe mixture with a chromatographic material comprising the porousmaterial, to thereby remove or isolate the component from the mixture.

In certain embodiments according the method of the invention, the porousmaterial is a poly(divinylbenzene-co-N-vinylcaprolactam) copolymer. Inanother certain aspect according to the method of the invention, thecomponent is a biological material. In yet another certain aspect, thebiological material is an intact protein, a denatured protein, amodified protein, an oligonucleotide, a modified oligonucleotide, asingle-stranded oligonucleotide, a double-stranded oligonucleotide, DNA,RNA, or a peptide. In particular aspect, the biological material is aninclusion body, a biological fluid, a biological tissue, a biologicalmatrix, an embedded tissue sample, or a cell culture supernatant.

In one embodiment, the present invention provides a method fordetermining the level of a component in a mixture, comprising:

-   -   a) contacting the mixture with a chromatographic material        comprising the porous material of the invention under conditions        that allow for sorption of the component onto the porous        materials;    -   b) washing the chromatographic material having the sorbed        component with a solvent under conditions so as to desorb the        component from the porous materials; and    -   c) determining the level of the desorbed component.

According to one embodiment of the invention, a separation devicecomprises the porous material of the invention. In certain embodiments,the separation device is selected from the group consisting ofchromatographic columns, cartridges, thin layer chromatographic plates,filtration membranes, sample clean up devices, solid phase organicsynthesis supports, and microtiter plates.

In related embodiments, the present invention provides a hydrophilicinteraction chromatography cartridge comprising the porous material. Incertain embodiments, the cartridge comprises an open-ended column thatcontains the porous material.

In one aspect of the invention, a method of analyzing a glycosylatedproteinaceuous sample comprises a step of contacting said sample with astationary phase material in a chromatography column wherein saidstationary phase material comprises a plurality of pores. In particularembodiments, the stationary phase material comprises at least onehydrophilic monomer and a poly-amide bonded phase.

In certain aspect, the glycosylated proteinaceuous sample is derivedfrom a glycoprotein or a glycosylated monoclonal antibody.

In another certain aspect, the stationary phase is fully porous orsuperficially porous. In related aspect, the average diameter of saidpores is greater than or equal to about 200 Å, greater than or equal toabout 250 Å, greater than or equal to about 300 Å, or greater than orequal to about 450 Å. In another related aspect, the average diameter ofsaid pores is from about 1 to about 50 Å, from about 5 to about 40 Å, orfrom about 10 to about 30 Å.

In another aspect of the invention, the stationary phase materialcomprises an organic-inorganic hybrid core comprising an aliphaticbridged silane. In related aspect, an aliphatic group of the aliphaticbridged silane is ethylene.

In certain aspect, the stationary phase material is in one or more formsof particles. In particular aspect, the average diameter of theparticles is from about 0.1 m and about 500 m, from about 1 m and about100 m, or from about 1 m and about 10 m. In another certain aspect, thestationary phase material further comprises a porous monolith.

According to the invention, the methods comprise a step of preparingsaid sample in a sample diluent. In certain aspect, an aqueous samplediluent has an injection volume of less than about 1/100th of thechromatography column volume.

In another certain aspect, the sample diluent comprises a denaturant. Inparticular aspect, the denaturant is guanidine hydrochloride (GuHCl).

In yet another aspect, the step of preparing the sample furthercomprises reduction, enzymatic digestion, denaturation, fragmentation,chemical cleavage and a combination thereof.

In certain aspect according to the method of the invention, a mobilephase for the chromatography is a high organic eluent. In a relatedaspect, the mobile phase for chromatography is one or more selected fromthe group consisting of acetonitrile, isopropanol, n-propanol, methanol,ethanol, butanol, water and a mixture thereof.

In certain aspect of the invention, the method comprises a step ofadding an ion pairing agent to the mobile phase eluent. In particularaspect, the ion pairing agent is selected from the group consisting oftrifluoroacetic acid, heptafluorobutyric acid, pentafluoropropionicacid, nonafluoropentanoic acid, acetic acid, propanoic acid, andbutanoic acid.

In another certain aspect of the method, a column pressure whencontacting the sample with the stationary phase is no less than about3,000 psi, no less than about 4,000 psi, no less than about 5,000 psi,no less than about 6,000 psi, or no less than about 7,000 psi.

According to the invention, the method comprises a step of identifyingthe glycopeptide. In particular aspect, the identification step isachieved by ultraviolet detection, ESI-MS, evaporative light scattering,fluorescence, mass spectrometry, MALDI-MS, ESI-MS, MALDI MS/MS, ESIMS/MS, MSn, MSe, nuclear magnetic resonance, infrared analysis or acombination thereof. In another particular aspect, the identification ofthe glycosylation of peptide is further achieved by comparison of massspectrometry peaks with known compounds in a computer database.

In another aspect of the invention, a method of performing hydrophilicchromatography (HILIC) for characterizing glycopeptide comprises stepsof:

-   -   a) preparing a sample containing the glycopeptide in a sample        diluent;    -   b) providing a column having an inlet and an outlet and a        stationary phase material in the column wherein the stationary        phase material comprises a plurality of pores;    -   c) loading the sample on the stationary phase material at a        column inlet pressure of no less than about 3,000 psi and        flowing the sample with a mobile phase eluent through said        column;    -   d) separating the sample from the outlet into one or more        fractions; and    -   e) identifying the fractions.

In particular embodiments, the stationary phase material comprises atleast one hydrophilic monomer and a poly-amide bonded phase.

In certain aspect, the glycopeptide is derived from a glycoprotein or aglycosylated monoclonal antibody.

In another certain aspect, the stationary phase material is fully porousor superficially porous. In related aspect, the average diameter of saidpores is greater than or equal to about 200 Å, greater than or equal toabout 250 Å, greater than or equal to about 300 Å, or greater than orequal to about 450 Å. In another related aspect, the average diameter ofsaid pores is from about 1 to about 50 Å, from about 5 to about 40 Å, orfrom about 10 to about 30 Å.

In another aspect of the invention, the stationary phase materialcomprises an organic-inorganic hybrid core comprising an aliphaticbridged silane. In related aspect, an aliphatic group of the aliphaticbridged silane is ethylene.

In certain aspect, the stationary phase material is in one or more formsof particles. In particular aspect, the average diameter of theparticles is from about 0.1 μm and about 500 μm, from about 1 μm andabout 100 μm, or from about 1 μm and about 10 μm. In another certainaspect, the stationary phase material further comprises a porousmonolith.

In certain aspect, an aqueous sample diluent has an injection volume ofless than about 1/100th of the column volume. In another certain aspect,the sample diluent comprises a denaturant. In particular aspect, thedenaturant is guanidine hydrochloride (GuHCl).

In yet another aspect, the step of preparing the sample is reduction,enzymatic digestion, denaturation, fragmentation, chemical cleavage or acombination thereof.

In certain aspect according to the method of the invention, the mobilephase for the chromatography is a high organic eluent. In a relatedaspect, the mobile phase for chromatography is one or more selected fromthe group consisting of acetonitrile, isopropanol, n-propanol, methanol,ethanol, butanol, water and a mixture thereof.

In certain aspect of the invention, the method comprises a step ofadding an ion pairing agent to the mobile phase eluent. In particularaspect, the ion pairing agent is selected from the group consisting oftrifluoroacetic acid, heptafluorobutyric acid, pentafluoropropionicacid, nonafluoropentanoic acid, acetic acid, propanoic acid, andbutanoic acid.

In certain embodiments, the mobile phase can include a gradient ofmobile phases such that a gradient in pH, salt concentration,temperature, or other parameter is established.

In another certain aspect of the method, the column inlet pressure whenloading the sample on the stationary phase material is no less thanabout 4,000 psi, no less than about 5,000 psi, no less than about 6,000psi, or no less than about 7,000 psi.

According to the invention, the method further comprises a step ofidentifying the glycopeptide. In particular aspect, the identificationstep is achieved by ultraviolet detection, ESI-MS, evaporative lightscattering, fluorescence, mass spectrometry, MALDI-MS, ESI-MS, MALDIMS/MS, ESI MS/MS, MSn, MSe, nuclear magnetic resonance, infraredanalysis or a combination thereof. In another particular aspect, theidentification of the glycosylation of peptide is further achieved bycomparison of mass spectrometry peaks with known compounds in a computerdatabase.

In particular embodiment, the present invention provides a method ofperforming hydrophilic interaction chromatography (HILIC) forcharacterizing a glycosylated monoclonal antibody comprising steps of:

-   -   a) preparing a sample containing the glycosylated monoclonal        antibody, wherein the glycopeptide is prepared by reduction,        enzymatic digestion, denaturation, fragmentation, chemical        cleavage or a combination thereof;    -   b) providing a column having an inlet and an outlet; and a        stationary phase material wherein said stationary phase material        comprises a plurality of pores having a size greater than or        equal to than about 300 Å or between about 1 Å and about 50 Å in        diameter;    -   c) loading the sample on said stationary phase material at the        column inlet pressure of no less than about 3,000 psi and        flowing the sample with a mobile phase eluent through said        chamber, wherein an ion paring agent is added to the sample        and/or eluent and the mobile phase eluent is selected from the        group consisting of acetonitrile, isopropanol, n-propanol,        methanol, ethanol, butanol, water and a mixture thereof, and        wherein an aqueous injection volume is less than about        1/100^(th) of a column volume;    -   d) separating one or more sample fractions;    -   e) identifying the fraction by ultraviolet detection, ESI-MS,        evaporative light scattering, fluorescence, mass spectrometry,        MALDI-MS, ESI-MS, MALDI MS/MS, ESI MS/MS, MS^(n), MS^(e),        nuclear magnetic resonance, infrared analysis or a combination        thereof, or by comparison of mass spectrometry peaks with known        compounds in a computer database.

In particular embodiments, the stationary phase material comprises atleast one hydrophilic monomer and a poly-amide bonded phase.

In another particular embodiment, the invention provides a method ofperforming hydrophilic chromatography (HILIC) for separation ofglycosylated proteins or peptides comprising steps of:

-   -   a) preparing a sample containing the glycosylated proteins or        peptides,    -   b) providing a HILIC column having an inlet and an outlet; and a        stationary phase material,    -   c) loading the sample on said stationary phase material at a        column inlet pressure of no less than about 3,000 psi and        flowing the sample with a mobile phase eluent through said        column,    -   d) separating the sample from the outlet into one or more        fractions.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other features of the present invention will now bedescribed in detail with reference to certain exemplary embodimentsthereof illustrated by the accompanying drawings which are given hereinbelow by way of illustration only, and thus are not limitative of thepresent invention.

FIG. 1 is a set of chromatograms comparing RNase B separations achievedwith different types of stationary phases, a 5 μm unbonded stationaryphase with 130 Å pores, a hydrophilic, poly-amide bonded stationaryphase with 130 Å pores, and a hydrophilic, poly-amide bonded stationaryphase with 300 Å pores (a wide-pore phase). (ACQUITY H-Class Bio, A214,2 Hz 2.1×150 mm, 0.2 mL/min Injection Volume: 0.5 μL; 1 μg Protein;Mobile Phase A: 0.1% TFA, H₂O; Mobile Phase B: 0.1% TFA, CAN; 20% to 80%H₂O in 20 min Column Temperature: 80° C.)

FIG. 2 is a set of chromatograms corresponding to RNase B separationsobtained with a shallow gradient to better demonstrate the performancedifferences between a hydrophilic, poly-amide bonded stationary phasewith 130 Å pores and a hydrophilic, poly-amide bonded stationary phasewith 300 Å pores (a wide-pore phase). Peak identifications:1—aglycosylated, 2—ManS, 3—Man6, 4—Man7, 5—Man8, 6—Man9. (ACQUITYH-Class Bio BEH Amide, 1.7 μm, 130/300 Å 2.1×150 mm, 1.7 μm; Flow Rate:0.1-0.4 mL/min; Column Temp.: 30° C.; Injection Volume: 0.5 μL; 1 μgProtein; Mobile Phase A: 0.1% TFA, H₂O; Mobile Phase B: 0.1% TFA, CAN;20% to 34% H₂O in 1 min, then 34% to 41% H₂O in 20 min)

FIG. 3 is a set of chromatograms comparing the RNase B separationsachieved with different types of ion pairing agents, 0.5% (v/v) formicacid (FA), 50 mM ammonium formate, and 0.1% (v/v) trifluoroacetic acid(TFA). (ACQUITY H-Class Bio, A280, 2 Hz BEH Amide, 1.7 μm, 300 Å 2.1×150mm, 0.2 mL/min; Injection Volume: 0.5 μL; 1 μg Protein; Mobile Phase A:0.1% TFA, H₂O; Mobile Phase B: CAN; 20% to 80% H₂O in 20 min ColumnTemperature: 80° C.)

FIGS. 4A-B are chromatograms and mass spectra demonstrating the effectof pressure on a separation of the glycoforms of intact trastuzumab.FIG. 4A shows MS based identifications of the trastuzumab glycoforms,including MS data (from RT 18-22 min) indicative of aggregate formationat a 3200 psi column pressure. FIG. 4B shows a set of chromatogramswherein post-column flow restriction was used to increase columnpressure from 3200 psi, to 4500 psi, and to 7300 psi. The highest columnpressure proved effective in minimizing the sample aggregation definedin FIG. 4A. (3 μg Protein; Temp.: 30° C.; Mobile Phase A: 0.1% TFA, H₂O;Mobile Phase B: 0.1% TFA, ACN; 20% to 30% H₂O in 1 min, then 30% to 37%H₂O in 20 min; ACQUITY H-Class Bio, A214, 2 Hz Xevo G2 Qtof, 500-4000m/z, 2 Hz; BEHAmide, 1.7 μm, 300 Å; 2.1×150 mm, 1.7 μm, 0.2 mL/min;Injection Volume: 1.5 μL)

FIG. 5 is chromatograms showing comparative view of separation of RNaseB of different aqueous sample loading volumes. (ACQUITY H-Class Bio BEHAmide, 1.7 μm, 300 Å 2.1×150 mm, 1.7 μm, 0.2 mL/min InjectionVolume:0.5-10 μL 1 μg RNase B; Temp.: 30° C.; Mobile Phase A: 0.1% TFA,H₂O; Mobile Phase B: 0.1% TFA, ACN; 20% to 34% H₂O in 1 min, then 34% to41% H₂O in 20 min)

FIG. 6 is a chromatogram showing comparative views of separation ofRNase B and intact trastuzumab at different column temperatures of theranges from 30° C. to 80° C. (ACQUITY H-Class Bio, A214, 2 Hz, BEHAmide, 1.7 μm, 300 Å 2.1×150 mm, 0.2 mL/min, Injection Volume: 0.5 μL; 1μg Protein; Mobile Phase A: 0.1% TFA, H₂O; Mobile Phase B: ACN; 20% to80% H₂O in 20 min)

FIG. 7 is a chromatogram showing comparative view of separation of RNaseB at different flow rates, 0.1 mL/min, 0.2 mL/min, 0.3 mL/min, and 0.4mL/min.

FIGS. 8A-B are chromatograms of IdeS-digested trastuzumab characterizedwith the stationary phase material in the invention. (ACQUITY H-ClassBio, A214, 2 Hz Xevo G2 QTof, 500-4000 m/z, 2 Hz; 2.1×150 mm, 0.2mL/min; Mobile Phase A: 0.1% TFA, H₂O; Mobile Phase B: 0.1% TFA, ACN; 1μg Protein; Injection Volume: 0.67 μL; BEH Amide, 1.7 μm, 300 Å′ Temp.:30° C.′ 20% to 30% H₂O in 1 min, then 30% to 37% H₂O in 20 min)

FIG. 9 is a chromatogram of Lys-C-digested trastuzumab characterizedwith the stationary phase material in the invention. (ACQUITY H-ClassBio A214 10 Hz Synapt G2-S, 50-2500 m/z, 10 Hz 2.1×150 mm, 0.2 mL/min′9.2 μg Lys-C Digested Trasutuzumab′ Mobile Phase A: 0.1% TFA, H₂O′Mobile Phase B: 0.1% TFA, ACN)

FIG. 10 is a comparative view of chromatograms corresponding totrastuzumab characterized with the stationary phase material in theinvention according to different denaturation/reduction procedures anddiluent conditions. (ACQUITY H-Class Bio, A214, 2 Hz Xevo G2 QTof,500-4000 m/z, 2 Hz BEH Amide, 1.7 m, 300 Å 2.1×150 mm, 1.7 m, 0.2 mL/minInjection Volume: 0.67 μL 1 μg; Protein Temp.: 30° C. Mobile Phase A:0.1% TFA, H₂O; Mobile Phase B: 0.1% TFA, ACN; 20% to 30% H₂O in 1 min,then 30% to 37% H₂O in 20 min)

FIG. 11 is a chromatogram showing orthogonality of separation between areversed phase C4 column and the hydrophilic, poly-amide bondedstationary phase column. RNase B is analyzed in this chromatogram.(ACQUITY H-Class Bio, A214, 2 Hz Xevo G2 QTof, 500-4000 m/z, 2 Hz2.1×150 mm, 0.2 mL/min Mobile Phase A: 0.1% TFA, H₂O; Mobile Phase B:0.1% TFA, ACN; 1 μg Protein Injection Volume: 0.67 μL/BEH300 C4, 1.7 m,300 Å Temp.: 80° C.; 5% to 33.3% ACN in 1 min, then 33.3% to 40.3% ACNin 20 min/BEH Amide, 1.7 m, 300 Å Temp.: 30° C.; 20% to 30% H₂O in 1min, then 30% to 37% H₂O in 20 min)

FIG. 12 is a set of chromatograms corresponding to Lys-C glycopeptidemapping of trastuzumab performed with a hydrophilic, poly-amide bondedstationary phase with 130 Å pores versus a hydrophilic, poly-amidebonded stationary phase with 300 Å pores (a wide-pore phase). Thedifference in the resolution (R_(s)) of the glycopeptide map is noted.(ACQUITY H-Class Bio BEH Amide, 1.7 m, 130 or 300 Å 2.1×150 mm, 1.7 m,0.2 mL/min; Temp.: 30° C.; 9.2 μg Lys-C Digested Trasutuzumab; MobilePhase A: 0.1% TFA, H₂O; Mobile Phase B: 0.1% TFA, ACN; 20% to 50% MPA in60 min A214, 10 Hz)

FIG. 13 presents chromatograms corresponding to Lys-C glycopeptidemapping of trastuzumab obtained with a hydrophilic, poly-amide bondedstationary phase with 300 Å pores versus analysis of anthranilamidelabeled, released N glycans (from trastuzumab) obtained with ahydrophilic, poly-amide bonded stationary phase with 130 Å pores.(ACQUITY H-Class Bio A214, 10 Hz Synapt G2-S, 50-2500 m/z, 10 Hz; BEHAmide, 1.7 m, 300 Å 2.1×150 mm, 1.7 m, 0.2 mL/min Temp.: 30° C. 9.2 μgLys-C Digested Trasutuzumab; Mobile Phase A: 0.1% TFA, H2O Mobile PhaseB: 0.1% TFA, ACN 20% to 50% MPA in 60 min/ACQUITY H-Class Bio FLR,360/428 nm, 10 Hz Synapt G2-S, 500-2500 m/z, 2 Hz; BEH Glycan/Amide, 1.7m, 130 Å 2.1×150 mm, 1.7 m, 0.4 mL/min Temp.: 40° C. 2-AB LabeledGlycans Released from 9.2 μg Trasutuzumab (GlycoWorks); Mobile Phase A:50 mM NH4Formate, H2O, pH 4.4; Mobile Phase B: ACN; 30% MPA for 2.06min, then 30% to 47% MPA over 32.74 min)

FIG. 14 shows a set of chromatograms corresponding to IdeS-digestedtrastuzumab from different manufactured batches as obtained with thestationary phase material in the invention. Integration and relativequantitation of the different, resolved glycoforms is demonstrated.(ACQUITY H-Class Bio, A214, 2 Hz Xevo G2 QTof, 500-4000 m/z, 2 Hz2.1×150 mm, 0.2 mL/min Mobile Phase A: 0.1% TFA, H₂O Mobile Phase B:0.1% TFA, ACN; 20% to 30% H2O in 1 min, then 30% to 37% H₂O in 20 min; 1μg Protein; Temp.: 30° C.; Injection Volume: 0.67 μL; BEH Amide, 1.7 m,300 Å)

FIG. 15 is a chromatogram corresponding to IdeS-digested,carboxypeptidase B treated cetuximab as obtained with the stationaryphase material in the invention. (ACQUITY H-Class Bio, A214, 2 Hz XevoG2 QTof, 500-4000 m/z, 2 Hz; 2.1×150 mm, 0.2 mL/min Mobile Phase A: 0.1%TFA, H2O Mobile Phase B: 0.1% TFA, ACN; 20% to 30% H2O in 1 min, then30% to 37% H₂O in 20 min; 1 μg Protein; Temp.: 60° C.; Injection Volume:0.67 μL; BEH Amide, 1.7 m, 300 Å)

FIG. 16 presents a chromatogram corresponding to tryptic glycopeptidemapping of trastuzumab obtained with a hydrophilic, poly-amide bondedstationary phase with 300 Å pores (the stationary phase material in theinvention). (ACQUITY H-Class Bio, A214, 210 Hz SynaptG2-S, 50-2500 m/z,10 Hz BEH Amide, 1.7 μm, 300 Å 2.1×150 mm, 1.7 m, 0.2 mL/min InjectionVolume: 243.2 μL Sample Diluent: 80% ACN; 9.2 μg Lys-C/Trypsin Digest ofReduced and Alkylated Cetuximab Temp.: 60° C. Mobile Phase A: 0.1% TFA,H₂O Mobile Phase B: 0.1% TFA, ACN 2)

FIG. 17 presents RNase B separations with amide bonded stationary phasesconstructed from different particle morphologies (Prototypes A-D). RNaseB (2 μg) separated at 30° C.

FIG. 18 presents RNase B Separations with 1.7 m amide bonded BEH (300 ÅAPD) bonded so as to have different N coverages (Prototypes E and F).Chromatographic resolution (peak width at half height) and pressures(system) are noted. RNase A (0.2 μg) and RNase B (1.8 μg) separated at45° C.

It should be understood that the appended drawings are not necessarilyto scale, presenting a somewhat simplified representation of variouspreferred features illustrative of the basic principles of theinvention. The specific design features of the present invention asdisclosed herein, including, for example, specific dimensions,orientations, locations, and shapes will be determined in part by theparticular intended application and use environment.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The present invention will be more fully illustrated by reference to thedefinitions set forth below.

The term “hydrophilic” describes having an affinity for, attracting,adsorbing or absorbing water.

The term “hydrophobic” describes lacking an affinity for, repelling, orfailing to adsorb or absorb water.

The term “ion-exchange functional group” is intended to include a groupwhere the counter-ion is partially free and can readily be exchanged forother ions of the same sign.

The term “mole percent” describes the mole fraction, expressed as apercent, of the monomer of interest relative to the total moles of thevarious (two or more) monomers that comprise the copolymer of the porousmaterial of the invention.

The term “monolith” is intended to include a porous, three-dimensionalmaterial having a continuous interconnected pore structure in a singlepiece. A monolith is prepared, for example, by casting precursors into amold of a desired shape. The term monolith is meant to be distinguishedfrom a collection of individual particles packed into a bed formation,in which the end product still comprises individual particles in bedformation.

The term “monomer” is intended to include a molecule comprising one ormore polymerizable functional groups prior to polymerization, or arepeating unit of a polymer.

The term “porous material” is intended to include a member of a class ofporous crosslinked polymers penetrated by pores through which solutionscan diffuse. Pores are regions between densely packed polymer chains. Incertain embodiments, pores include vacant space which is usually definedwith a size of diameter. A pore on the chromatographic surface isusually open-ended so the molecule smaller than the size of the pore canreside in or pass through the pore and can be in any forms. Becausepores in plurality provide large surface area as well as an alternativeflow path, pores on the chromatographic surface can impact on retentiontime of analytes in chromatography and give chromatographic enhancementsincluding high separation efficiency and good mass transfer properties(as evidenced by, e.g., reduced band spreading and good peak shape).Thus, the size distribution of pores usually is a key contributor forthe benefit of the chromatography in the invention. Pores ofchromatographic surface can be introduced with a synthesized polymer,for example, an ethylene bridged hybrid (BEH Technology™, WatersCorporation, Milford, Mass.,) with a silica that create an inertchemical structure.

The term “random ordering” is intended to include ordering in whichindividual units are joined randomly.

The term “hydrophilic interaction chromatography” or HILIC is intendedto include a process employing a hydrophilic stationary phase and ahydrophobic organic mobile phase in which hydrophilic compounds areretained longer than hydrophobic compounds. In certain embodiments, theprocess utilizes a water-miscible solvent mobile phase. In certainembodiments, the term also includes ERLIC (electrostatic repulsionhydrophilic interaction chromatography), Cationic ERLIC and AnionicERLIC.

The term “sorption” describes the ability of a material to take up andhold another material by absorption or adsorption.

The term “surface modifiers” includes (typically) functional groupswhich impart a certain chromatographic functionality to the material.

The language “surface modified” is used herein to describe the compositematerial of the present invention that possess organic groups which mayadditionally be substituted or derivatized with a surface modifier.“Surface modifiers” include, but are not limited to, organic functionalgroups that impart a certain chromatographic functionality to thematerial.

The language “surface functionalized” is used herein to describe thecomposite material of the present invention that posses ion-exchangefunctional groups that impart a certain chromatographic functionality tothe material.

The term as used herein, “sample” refers to a mixture of molecules thatcomprises at least an analyte molecule, e.g., glycoprotein, that issubjected to manipulation in accordance with the methods of theinvention, including separating, analyzing, extracting, concentrating orprofiling.

The term as used herein, “analysis” or “analyzing” are usedinterchangeably and refer to any of the various methods of separating,detecting, isolating, purifying, solubilizing, detecting and/orcharacterizing molecules of interest (e.g., glycoprotein). Examplesinclude, but are not limited to, solid phase extraction, solid phasemicro extraction, electrophoresis, mass spectrometry, e.g., HILIC,MALDI-MS or ESI, liquid chromatography, e.g., high performance, e.g.,reverse phase, normal phase, or size exclusion, ion-pair liquidchromatography, liquid-liquid extraction, e.g., accelerated fluidextraction, supercritical fluid extraction, microwave-assistedextraction, membrane extraction, soxhlet extraction, precipitation,clarification, electrochemical detection, staining, elemental analysis,Edmund degradation, nuclear magnetic resonance, infrared analysis, flowinjection analysis, capillary electrochromatography, ultravioletdetection, and combinations thereof.

The term as used herein, “profiling” refers to any of various methods ofanalysis which are used in combination to provide the content,composition, or characteristic ratio of biological molecules (e.g.,glycoprotein) in a sample.

The term as used herein, “chromatographic surface” includes a surfacewhich is exposed to a sample or analytes. Chromatographic surface can bechemically modified, functionalized or activated or have amicrostructure, e.g. a pore. In certain embodiments, the chromatographicsurface can be hydrophobic, hydrophilic (polar) or ionic. In otherembodiments, the chromatographic surface is fully porous, superficiallyporous or non-porous.

The term as used herein, “chromatographic core” includes achromatographic material, including but not limited to an organicmaterial such as silica or a hybrid material, as defined herein, in theform of a particle, a monolith or another suitable structure which formsan internal portion of the materials of the invention. In certainaspects, the surface of the chromatographic core represents thechromatographic surface, as defined herein, or represents a materialencased by a chromatographic surface, as defined herein. Thechromatographic surface material may be disposed on or bonded to orannealed to the chromatographic core in such a way that a discrete ordistinct transition is discernible or may be bound to thechromatographic core in such a way as to blend with the surface of thechromatographic core resulting in a gradation of materials and nodiscrete internal core surface. In certain aspects, the chromatographicsurface material may be the same or different from the material of thechromatographic core and may exhibit different physical orphysiochemical properties from the chromatographic core, including, butnot limited to, pore volume, surface area, average pore diameter, carboncontent or hydrolytic pH stability.

The term as used herein, “amide” is intended to include a derivativeform of carboxylic acid in which the hydroxyl group has been replaced byan amine or ammonia. Due to the existence of strong electronegativeatoms, oxygen and nitrogen, next to carbon, dipole moment is producedand the molecule with amide group presents polarity or hydrophilicity.In certain aspects of chromatography, amide group may be covalentlybonded to the surface of the chromatographic core to impart ahydrophilicity to a chromatographic stationary phase.

The term as used herein, “hybrid”, including “hybrid inorganic/organicmaterial,” includes inorganic-based structures wherein an organicfunctionality is integral to both the internal or “skeletal” inorganicstructure as well as the hybrid material surface. The inorganic portionof the hybrid material may be, e.g., alumina, silica, titanium, cerium,or zirconium or oxides thereof, or ceramic material. “Hybrid” includesinorganic-based structures wherein an organic functionality is integralto both the internal or “skeletal” inorganic structure as well as thehybrid material surface.

The term as used herein, “ion pairing agent” is intended to include anionic compound that imparts a certain hydrophobicity to other molecule,e.g. an analyte. Ion paring agent includes, but is not limited to, ahydrophobic counterion, for example, trifluoroacetic acid (TFA). Ionparing agent is capable of ion-pairing with the positively chargedresidues of analytes, such as positively charged amino acid residues ofa peptide. Generally, ion pairing agent is added to the chromatographyto reduce hydrophilicity or enhance hydrophobicity of the analytemolecules.

The term as used herein, “glycopeptide/glycoprotein” is a modifiedpeptide/protein, during or after their synthesis, with covalently bondedcarbohydrates or glycan.

The term as used herein, “glycan” is a compound comprising one or moreof sugar units which commonly include glucose (Glc), galactose (Gal),mannose (Man), fucose (Fuc), N-acetylgalactosamine (GalNAc),N-acetylglucosamine (GlcNAc) and N-acetylneuraminic acid (NeuNAc) (FrankKjeldsen, et al. Anal. Chem. 2003, 75, 2355-2361). The glycan moiety inglycoprotein is an important character to identify its function orcellular location. For example, most membrane bound proteins areglycoproteins for their intercellular or extracellular function. Inother examples, a specific monoclonal antibody, e.g., trastuzumab (acommercial monoclonal antibody used for breast cancer treatment), ismodified with specific glycan moiety.

Compositions of the Invention

In one aspect, the invention provides, a porous material comprising acopolymer comprising at least one hydrophilic monomer and a poly-amidebonded phase, wherein the average pore diameter is greater than or equalto about 200 Å. In certain embodiments, the average pore diameter isgreater than or equal to about 250 Å. In still other embodiments, theaverage pore diameter is greater than or equal to about 300 Å. In stillanother embodiments, the average pore diameter is greater than or equalto about 450 Å.

In another aspect, the invention provides a porous material comprising acopolymer comprising at least one hydrophilic monomer and a poly-amidebonded phase, wherein said material has a median pore diameter of about100 Å to about 1000 Å. Median pore diameter can be measured, forexample, by inverse size exclusion chromatography (I-SEC). In certainaspects, the material has a median pore diameter of about 200 Å to about800 Å; about 300 Å to about 550 Å; about 100 Å; about 200 Å; about 300Å; about 400 Å; about 425; about 450 Å; about 475 Å; about 500 Å; about525 Å; about 550 Å; about 575 Å; about 600 Å; about 700 Å; or about 800Å.

The invention further provides a porous material comprising a copolymercomprising at least one hydrophilic monomer and a poly-amide bondedphase, wherein said material has nitrogen content from about 0.5% N toabout 20% N; from about 1% N to about 10% N; from about 1% N to about 5%N; from about 1% N to about 4% N; about 1% N; about 1.5% N; about 2% N;about 2.5% N; about 3% N; about 3.5% N; about 4% N; about 4.5% N; about5% N; about 5.5% N; about 6% N; about 6.5% N; about 7% N; about 7.5% N;about 8% N; about 8.5% N; about 9% N; about 9.5% N; about 10% N; about10.5% N; about 11% N; about 11.5% N; about 12% N; about 12.5% N; about13% N; about 13.5% N; about 14% N; about 14.5% N; or about 15% N

In certain embodiments, the porous material of the invention has both amedian pore diameter of about 100 Å to about 1000 Å; about 200 Å toabout 900 Å; about 300 Å to about 800 Å; or about 300 Å to about 550 Å;and a nitrogen content from about 0.5% N to about 20% N; from about 1% Nto about 10% N; from about 1% N to about 5% N; from about 1% N to about4% N; about 1% N; about 1.5% N; about 2% N; about 2.5% N; about 3% N;about 3.5% N; about 4% N; about 4.5% N; about 5% N; about 5.5% N; about6% N; about 6.5% N; about 7% N; about 7.5% N; about 8% N; about 8.5% N;about 9% N; about 9.5% N; about 10% N; about 10.5% N; about 11% N; about11.5% N; about 12% N; about 12.5% N; about 13% N; about 13.5% N; about14% N; about 14.5% N; or about 15% N.

In other embodiments, the porous material comprising a copolymercomprising at least one hydrophilic monomer and a poly-amide bondedphase, wherein said material has an oxygen content from about 1% O toabout 20% O; from about 1% O to about 10% O; from about 1% O to about 5%O; from about 1% O to about 4% O; about 1% O; about 2% O; about 3% O;about 4% O; about 5% O; about 6% O; about 7% O; about 8% O; about 9% O;about 10% O; about 11% O; about 12% O; about 13% O; about 14% O; orabout 15% O.

In yet other embodiments, the porous material comprising a copolymercomprising at least one hydrophilic monomer and a poly-amide bondedphase, wherein said material has a sulfur content from about 1% S toabout 20% S; from about 1% S to about 10% S; from about 1% S to about 5%S; from about 1% S to about 4% S; about 1% S; about 2% S; about 3% S;about 4% S; about 5% S; about 6% S; about 7% S; about 8% S; about 9% S;about 10% S; about 11% S; about 12% S; about 13% S; about 14% S; orabout 15% S.

In still other embodiments, the porous material comprising a copolymercomprising at least one hydrophilic monomer and a poly-amide bondedphase, wherein said material has a phosphorous content from about 1% Pto about 20% P; from about 1% P to about 10% P; from about 1% P to about5% P; from about 1% P to about 4% P; about 1% P; about 2% P; about 3% P;about 4% P; about 5% P; about 6% P; about 7% P; about 8% P; about 9% P;about 10% P; about 11% P; about 12% P; about 13% P; about 14% P; orabout 15% P.

In certain aspects, the porous material has a specific surface area inthe range from about 50 to about 850 square meters per gram and poreshaving a diameter ranging from about 1 to 50 Å or from 200 to 1000 Å.

In certain embodiments, the porous materials of the invention take theform of porous particles, e.g., beads, pellets, or any other formdesirable for use. The porous particles can have, e.g., a sphericalshape, a regular shape or an irregular shape. In some embodiments, theparticles are beads having a diameter in the range from about 0.1 toabout 500 μm, from about 1 to about 100 μm, or from about 1 to about 10μm.

In other embodiments, the porous materials of the invention take theform of porous monoliths. In certain embodiments, the monoliths have thefollowing characteristics: surface area ranging from about 50 to about800 m²/g, more particularly about 300 to about 700 m²/g; pore volumeranging from about 0.2 to about 2.5 cm³/g, more particularly about 0.4to about 2.0 cm³/g, still more particularly about 0.6 to about 1.4cm³/g; and pore diameter ranging from about 20 to about 500 Å, moreparticularly about 50 to 300 Å, still more particularly about 80 toabout 150 Å.

Component Materials of the Invention

The porous materials of the invention comprise a copolymer a copolymercomprising at least one hydrophilic monomer and a poly-amide bondedphase. In certain embodiments, the copolymer of the invention isnon-sulfonated. In certain other embodiments, the copolymer issulfonated.

Hydrophilic Monomers

In certain embodiments, the hydrophilic monomer is3-methacryloxypropyltrichlorosilane,3-methacryloxypropylmethyldichlorosilane,3-methacryloxypropyldimethylchlorosilane,3-methacryloxypropyltrimethoxysilane,3-methacryloxypropylmethyldimethoxysilane,3-methacryloxypropyldimethylmethoxysilane,3-methacryloxypropyltriethoxysilane,3-methacryloxypropylmethyldiethoxysilane,3-methacryloxypropyldimethylethoxysilane,3-acryloxypropyltrichlorosilane, 3-acryloxypropylmethyldichlorosilane,3-acryloxypropyldimethylchlorosilane 3-acryloxypropyltrimethoxysilane,3-acryloxypropylmethyldimethoxysilane,3-acryloxypropyldimethylmethoxysilane, 3-acryloxypropyltriethoxysilane,3-acryloxypropylmethyldiethoxysilane,3-acryloxypropyldimethylethoxysilane, styrylethyltrichlorosilane,styrylethylmethyldichlorosilane, styrylethyldimethylchlorosilane,styrylethyltrimethoxysilane, styrylethylmethyldimethoxysilane,styrylethyldimethylmethoxysilane, styrylethyltriethoxysilane,styrylethylmethyldiethoxysilane, styrylethyldimethylethoxysilane,vinyltriethoxysilane, vinyltrimethoxysilane,N-(3-acryloxy-2-hydroxypropyl)-3-aminopropyl triethoxysilane,(3-acryloxypropyl) trimethoxysilane,O-(methacryloxyethyl)-N-(triethoxysilylpropyl)urethane,N-(3-methacryloxy-2-hydroxypropyl)-3-aminopropyl triethoxysilane,methacryloxy methyltriethoxysilane, methacryloxymethyl trimethoxysilane,methacryloxypropy methyldiethoxysilane, methacryloxypropylmethyldimethoxysilane, methacryl oxypropyltris (methoxyethoxy)silane,3-(N-styrylmethyl-2-aminoethylamino) propyltrimethoxysilanehydrochloride,

Poly-Amide Bonded Phase

In certain embodiments, poly-amide bonded phase is derived fromacrylamide, divinylbenzene, styrene, ethylene glycol dimethacrylate,1-vinyl-2-pyrrolidinone and tert-butylmethacrylate, acrylamide,methacrylamide, N,N′-(1,2-dihydroxyethylene)bisacrylamide,N,N′-ethylenebisacrylamide, N,N′-methylenebisacrylamide, butyl acrylate,ethyl acrylate, methyl acrylate, 2-(acryloxy)-2-hydroxypropylmethacrylate, N,N-bis(2-cyanoethyl)acrylamide,N-acryloyltris(hydroxymethyl)aminomethane, 3-(acryloxy)-2-hydroxypropylmethacrylate, trimethylolpropane triacrylate, trimethylolpropaneethoxylate triacrylate, tris[(2-acryloyloxy)ethyl] isocyanurate,acrylonitrile, methacrylonitrile, itaconic acid, methacrylic acid,trimethylsilylmethacrylate, N-[tris(hydroxymethyl)methyl]acrylamide,(3-acrylamidopropyl)trimethylammonium chloride,[3-(methacryloylamino)propyl] dimethyl(3-sulfopropyl)ammonium hydroxideinner salt,

In other embodiments, the materials of the invention comprise two ormore different poly-amide bonded phases. In particular embodiments, thematerials of the invention comprise a first and second poly-amide bondedphase.

In some embodiments, the second poly-amide bonded phase is derived fromN,N-methylenebisacrylamide, N,N-ethylenebisacrylamide,N,N-propylenebisacrylamide, N,N-butylenebisacrylamide,N,N′-(1,2-dihydroxyethylene)bisacrylamide, or1,4-bis(acryloyl)piperazine.

In particular embodiments in which the materials of the inventioncomprise a first and second poly-amide bonded phase, the firstpoly-amide bonded phase is present in about 35 to about 99 mole % of thetotal poly-amide bonded phases. In certain embodiments, the firstpoly-amide bonded phase is present in about 35 mole %, about 40 mole %,about 45 mole %, about 50 mole %, about 55 mole %, about 60 mole %,about 65 mole %, about 70 mole %, about 75 mole %, about 80 mole %,about 85 mole %, about 90 mole %, about 95 mole %, about 96 mole %,about 97 mole %, about 98 mole %, or about 99 mole % of the totalpoly-amide bonded phases. In other embodiments in which the materials ofthe invention comprise a first and second poly-amide bonded phase,second poly-amide bonded phase is present in 65 to 1 mole % of the totalpoly-amide bonded phases. In certain embodiments, the second poly-amidebonded phase is present in about 65 mole %, about 60 mole %, about 55mole %, about 50 mole %, about 45 mole %, about 40 mole %, about 35 mole%, about 30 mole %, about 25 mole %, about 20 mole %, about 15 mole %,about 10 mole %, about 5 mole %, about 4 mole %, about 3 mole %, about 2mole %, or about 1 mole % of the total poly-amide bonded phases.

Surface Functionalization/Modification

The porous materials, in either porous particle or monolith form, may befunctionalized to provide an ion-exchange functional moiety.

In certain embodiments, the ion-exchange functional moiety can be formedby formation of an amine functionality on materials of the inventionafter cholomethylation as in the methods described in U.S. Pat. No.7,731,844, which is incorporated herein by reference. In otherembodiments, an amine functionality can be formed by direct reactionwith a neat amine.

In accordance with the invention, the ion-exchange functional moiety canbe formed from a substituted acyclic amine or a substituted cyclicamine. The substitution can be at any of the ring atoms, includingheteroatoms. For example, in certain embodiments, the ion-exchangefunctional moiety is a substituted cyclic secondary amine, e.g.,N-methyldiazinane and 4-methylpiperidine.

In other embodiments, the aforesaid amines are advantageouslysubstituted by an electron withdrawing group. In certain embodiments,the electron withdrawing group is selected from the group consisting ofhalogens, aromatic groups, unsaturated groups, ethers, thioethers,nitriles, nitro groups, esters, amides, carbamates, ureas, carbonates,sulfonamides, sulfones, sulfoxides and heteroatoms, e.g., N, O and S. Incertain embodiments, the electron withdrawing group is a halogen, anether, or an aromatic group.

In accordance with the invention, the electron withdrawing group of theamine has the effect of lowering the average pK_(a) of the conjugateacid of the amine as compared to the conjugate acid of the amine withoutthe electron withdrawing group. In certain embodiments, the pK_(a)ranges from about 5 to about 7.

In certain embodiments, the acyclic amine substituted with an electronwithdrawing group includes benzylamine, N-methylbenzylamine,N-ethylbenzylamine, N-propylbenzylamine, N-butylbenzylamine,N-pentylbenzylamine, N-hexylbenzylamine, N-heptylbenzylamine,N-octylbenzylamine, N-nonylbenzylamine, N-decylbenzylamine,N-undecylbenzylamine, N-dodecylbenzylamine, N-tridecylbenzylamine,N-tetradecylbenzylamine, N-pentadecylbenzylamine,N-hexadecylbenzylamine, N-heptadecylbenzylamine, N-octadecylbenzylamine,dibenzylamine, aniline, N-methylaniline, N-ethylaniline,N-propylaniline, N-butylaniline, N-pentylaniline, N-hexylaniline,N-heptylaniline, N-octylaniline, N-nonylaniline, N-decylaniline,N-undecylaniline, N-dodecylaniline, N-tridecylaniline,N-tetradecylaniline, N-pentadecylaniline, N-hexadecylaniline,N-heptadecylaniline, N-octadecylaniline,bis(2,2,2-trifluoromethyl)amine, phenethylamine, N-methylphenethylamine,4-methylphenethylamine, 3-phenylpropylamine,1-methyl-3-phenylpropylamine, N-isopropylbenzylamine, and4-phenylbutylamine. In certain preferred embodiments, the acyclic aminesubstituted with an electron withdrawing group is benzylamine,N-methylbenzylamine, or phenethylamine. In a preferred embodiment, theacyclic amine substituted with an electron withdrawing group isN-methylbenzylamine.

In other embodiments, cyclic secondary amines substituted with anelectron withdrawing group include oxazetane, oxazolane, oxazinane,oxazepane, oxazocane, oxazonane, oxazecane, thiazetane, thiazolane,thiazinane, thiazepane, thiazocane, thiazonane, and thiazecane. In oneembodiment, the cyclic secondary amine is 1,4-oxazinane. In theseembodiments, one of ordinary skill in the art will appreciate that theelectron withdrawing group is a second heteroatom that has substitutedfor a carbon atom in the ring. For example, the ring carbon adjacent tothe nitrogen atom in azetidine is substituted by an oxygen to yieldoxazetane, an amine encompassed by the term “cyclic secondary aminesubstituted with an electron withdrawing group”.

In still other embodiments, an ion-exchange functional moiety can beformed by reaction of the materials of the invention with hydrogenperoxide.

In certain embodiments, surface functionalization can be attained on thematerials of the invention by the methods described in U.S. Pat. No.7,232,520 and U.S. Pat. No. 7,731,844, which are incorporated herein byreference.

In another embodiment, the materials of the invention may be surfacemodified by coating with a polymer.

In still another embodiment, the materials of the invention may besurface modified by a combination of organic group modification andcoating with a polymer. In a further embodiment, the organic groupcomprises a chiral moiety.

In other embodiments, the materials of the invention may be surfacemodified via formation of an organic covalent bond between an organicgroup on the material and the modifying reagent.

Grafted Materials

In certain embodiments, the porous materials of the invention comprise aporous or non-porous core, including, but not limited to an inorganiccore, an organic core or a hybrid core onto which a copolymer comprisingat least one hydrophilic monomer and a poly-amide bonded phase isgrafted. In certain other embodiments, the porous materials of theinvention comprise a polymeric, porous core made from at least onehydrophobic monomer onto which a polymer made from a least onehydrophilic monomer is grafted. In still other embodiments, the porousmaterials of the invention comprise a polymeric, porous core made fromat least one hydrophilic monomer onto which a polymer made from a leastone hydrophobic monomer is grafted.

In such embodiments, the hydrophilic monomers may be as describedherein. The cores may include a silica material; a hybridinorganic/organic material; a superficially porous material; or asuperficially porous particle.

Methods of Preparation

The porous materials of the invention can be prepared via a number ofprocesses and mechanisms including, but not limited to, chain additionand step condensation processes, radical, anionic, cationic,ring-opening, group transfer, metathesis, and photochemical mechanisms.

The copolymer can be prepared via standard synthetic methods known tothose skilled in the art, e.g., as described in the examples.

Furthermore, porous material may be produced by known methods, such asthose methods described in, for example, in U.S. Pat. Nos. 4,017,528;6,528,167; 6,686,035; 7,175,913; 7,731,844 and WO2004/041398.

Methods of the Invention: HILIC Chromatography

The present invention provides the facilitated characterization ofprotein glycosylation through novel HILIC separation methods andpreviously unused column packing materials.

In typical methods, glycan characterization relies on releasing glycansfrom peptide chains and then analyzing them separately from thepeptides. Because glycans are not suitable for UV detection, thereleased glycans are often labeled with a fluorophore tag, e.g.anthranilamide, anthranilic acid or 2-aminopyridine, for fluorescencedetection or a molecule, e.g. procainamide, with significant basicity soas to enable MS detection. However, this approach to glycan analysisonly gives a global assessment of glycosylation. In particular,site-specific information about glycans is lost due to this workflowrelying on a release procedure.

In one embodiment, the present invention provides improved results ofseparation and analysis upon the ability to study glycans while they arestill covalently linked to their counterpart proteins, proteinfragments, and peptides. In another embodiment, the present inventionprovides a novel chromatography method for profiling glycosylationoutlines at both the intact protein and peptide-levels of analysis. In apeptide-level analysis of glycoforms, there is also the benefit in abiopharmaceutical characterization that a single sample can be utilizedfor reversed phase peptide mapping, e.g. a Lys-C digest, and HILIC-basedglycopeptide mapping. Moreover, preserving the linkage between theglycan and peptide/protein facilitates the UV and MS detection based onthe proteinaceous component containing chromophores and basic residues.

In certain embodiments, problems related to glycoprotein, glycofragment,and glycopeptide HILIC separations also have been solved with thepresent invention. For instance, on-column aggregation of proteinsamples, low sensitivity of chromatographic detection of the glycanmoieties, and low resolution of peaks due to restricted pore diffusionand long intra/inter-particle diffusion distances.

In one embodiment of the invention, the invention provides methods ofanalyzing a glycopeptide in a sample, comprising a step of contactingsaid sample with a stationary phase wherein said stationary phasecomprises at least one hydrophilic monomer and a poly-amide bonded phasewith a plurality of pores, wherein the average diameter of the pores isgreater than or equal to about 200 Å, greater than or equal to about 250Å, greater than or equal to about 300 Å, or greater than or equal toabout 450 Å.

In another certain embodiments, the stationary phase has an average porediameter small enough in diameter to completely restrict access ofglycoproteins/glycopeptides from the pore network of the particle,wherein the average diameter of the pores is from 1 to about 50 Å, fromabout 5 to about 40 Å, or from about 10 to about 30 Å.

In particular, the methods include such improvements by elevating columnpressure to minimize/eliminate protein aggregation on-column, byemploying low volume injections of aqueous samples to improve ease ofuse, by injecting sample from diluents containing high concentrations ofdenaturant to limit self-association of protein chains, and by applyingan ion pairing agent as a mobile phase additive to reduce retention dueto proteinaceous analyte components and thereby increase the selectivityof separation toward the covalently attached glycan.

Typically, characterization of glycan moieties from the protein israther complicated. For example, glycans are released and analyzedseparately from the protein. However, glycan moieties cannot besubjected to UV detection and require additional tagging, e.g.anthranilamide, anthranilic acid, or 2-aminopyridine, for suitabledetection. Moreover, releasing such glycan moiety from the protein maylose the intact information of protein profiling.

Furthermore, the present invention provides efficient HILIC methods forseparations of large biomolecules with a novel packing material inchromatographic devices.

In certain embodiments, the invention provides HILIC Separation of largebiomolecules, glycosylated proteins or peptides, and the like, in whichthe column inlet pressure is no less than 3,000 psi (to minimize proteinaggregation). In certain embodiments, the column inlet pressure can beno less than 3000 psi, 4000 psi, 5000 psi, 6000 psi, or 7000 psi.

In certain embodiments, the high purity stationary phase materials ofthe invention can be applied to other separation methods such as HPLC,UPLC, or any of the methods described in Encyclopedia of AnalyticalChemistry, ed. Myers, John Wiley & Sons; for example in the chapterdescribed by Irgum, K. 2006. High-Performance Liquid Chromatography ofBiological Macromolecules. Encyclopedia of Analytical Chemistry; or inU. D. Neue, in HPLC Columns Theory, Technology and Practice, (John Wiley& Sons, Hoboken, N.J., 1997); or L. R. Snyder, J. J. Kirkland, J. W.Dolan in Introduction to Modern Liquid Chromatography, (John Wiley &Sons, Hoboken, N.J., 2010)

A. Preparation of Samples Containing Glycosylated, ProteinaceousCompounds

A glycosylated, proteinaceous compound in the invention can be, but isnot limited to, a glycoprotein, glycosylated monoclonal antibody, orglycopeptide.

For the method of HILIC chromatography of the invention, theglycosylated protein can be analyzed as intact protein, or be preparedby reduction, enzymatic digestion, denaturation, fragmentation, chemicalcleavage and a combination thereof.

Reduction is to reduce disulfide bonds into two thiols in a3-dimensional protein structure. Reduction can be performed byheat-denaturing, adding a surfactant, or adding a denaturing agent,e.g., guanidine.HCl (6M), in the presence of a reducing agent, e.g.TCEP. Enzymatic degradation is a digestion of the protein with aprotease, e.g., Achromobacter protease I (Lys-C) or trypsin. Inaddition, the glycoprotein can be denatured by heat or chemicals, orcombination thereof. Fragmentation is cleaving protein portions of asingle or multi-subunit protein, such as a monoclonal antibody, withphysical, biological or chemical method. For example, immunoglobulindegrading enzyme from S. pyogenes (IdeS) is commonly used for antibodysubunit fragmentation.

In one embodiment, the glycopeptide in a sample can be treated andprepared by reduction, enzymatic degradation, denaturation orfragmentation prior to contacting with the HILIC stationary phase inorder to increase selectivity or a chromatographic efficiency.

In particular embodiments, the sample can be prepared in a samplediluent containing high concentrations of denaturants to limitself-association of protein chains. In particular embodiments, thesample diluent has the compatible dielectric strength of the initialmobile phase composition. In another certain embodiments, the samplediluent comprises a denaturant. The denaturant can be, withoutlimitation, guanidine hydrochloride (GuHCl). As evidence, FIG. 10 showsthe effect of such denaturant in sample diluents according to differentdenaturation/reduction procedures and diluent conditions.

B. Stationary Phase

Stationary phase of chromatography mostly imparts the character tochromatography based on the mechanism of capturing the analytes ofinterest.

The chromatographic surface on the stationary phases can be bonded,without limitation, with a specific chemical group which is identifiedas a hydrophobic, ionic, polar, hydrophilic or in combination thereof.In one embodiment of the invention, the chromatographic surface ishydrophilic or polar. In other embodiments, the chromatographic surfaceis an amide bonded phase which imparts a polarity and hydrophilicity tohold the polar analytes. Meanwhile, the amide bonded phase can also becompatible with a reverse phase chromatography mobile phase.

The stationary phase may be constructed to produce desirable resolutionbetween aglycosylated and glycosylated isoforms of proteins, forinstance RNase A from the Mannose-5 species of RNAse B. Nitrogen (N)coverage can be altered as a means to modulate chromatographicperformance, as evidenced in FIG. 18. In a particular embodiment, the Ncoverage of the stationary phase is controlled so as to producechromatographic resolution (half-height peak width) greater than orequal to 18 for the separation of RNase A and RNase B (Mannose-5species) under the conditions outlined in Example 22. Otherchromatographic attributes can likewise be modulated using changes to Ncoverage. FIG. 18 highlights how different chromatographic pressures, inparticular chromatographic pressures under aqueous mobile phaseconditions, are obtained with stationary phases exhibiting different % Ncontent and different nitrogen (N) coverages.

Stationary phase can include, without limitation, pores fully orsuperficially in the chromatographic material. The size of the porestypically can limit the retention time of the analytes which may or maynot penetrate the pores during the chromatography. For example, theanalytes which is smaller than the size of the pore entrance flow viapores and retention time of that analytes increases. In certain,embodiments of the invention, the stationary phase includes a pluralityof pores, wherein the average diameter of the pores is greater than 200Å in diameter, 250 Å in diameter, 300 Å in diameter or 450 Å indiameter. In other embodiments of the invention, the stationary phase isfully or superficially porous. As evidenced in FIGS. 1-2 and FIG. 12,the pore size larger than 200 Å further provides chromatographicenhancements including high separation efficiency and good mass transferproperties. Moreover, the exemplary chromatography provided in FIG. 17(Prototypes C and D) shows evidence of the utility of wide pore (>200 AAPD), amide bonded superficially porous stationary phases.

While not limited to theory, the amide bonded phase purposed forefficient glycoprotein and glycopeptide may be constructed of a fullyporous or superficially porous morphology, wherein the pore diameter issmall enough in diameter to completely restrict access ofglycoproteins/glycopeptides from the pore network of the particle. Theadsorption, partitioning and diffusion would thereby be limited to theparticle surface, such that band broadening due to restricted diffusionand long diffusion distances would be minimized. In certain embodiments,pore diameters of <50 Å can be used to achieve such an effect. (FIG. 17,Prototype A). In other certain embodiments, the average diameter of thepores is from 1 to about 50 Å, from about 5 to about 40 Å, or from about10 to about 30 Å. Particles with this morphology are predicted tofunction as a non-porous particle but to exhibit a surface with a higherco-efficient of friction than particles that are genuinely non-porous.This feature is predicted to enhance the packing and stability of thephase once packed into a column.

In certain embodiments, the stationary phase material is in one or moreforms of particles. The average diameter of the particles is from about0.1 μm and about 500 μm, from about 1 μm and about 100 μm, or from about1 μm and about 10 μm. In another certain embodiments, the stationaryphase material further comprises a porous mothlith.

The material used for the stationary phase may include a chromatographiccore. Chromatographic core material may interact with the analytes ofinterest or may not be involved in such interaction. Chromatographiccore may be an organic-inorganic hybrid core comprising an aliphaticbridged silane. In certain other specific embodiments, the aliphaticgroup of the aliphatic bridged silane is ethylene. The ethylene bridgedhybrid (BEH Technology™, Waters Corporation, Milford, Mass., hereafterBEH) silica imparts porosity to the stationary phase and the size of thepores is determined by the BEH.

In certain embodiments, the methods of the invention use BEH HILICcolumns (Waters Corporation, Milford Mass.) as a sample analysis tool.The BEH HILIC columns are porous, capable of containing various pores ofdifferent sizes and maintain high retention and capacity for polaranalytes, especially when the mobile phase is a reversed phase solvent.

For example, the ACQUITY UPLC™ BEH HILIC (Waters Corporation, Milford,Mass.) and ACQUITY UPLC™ BEH Amide columns (Waters Corporation, Milford,Mass.) featuring a mixed-mode retention mechanism (hydrophilic andsize-exclusion) can be modified very predictably for maximum selectivityand sensitivity.

It has been appreciated that the BEH HILIC and BEH Amide columns providea range of options for method development in the invention. These uniquecolumns are optimized and tested to produce efficient, reproducibleseparations under HPLC, UHPLC, and UPLC HILIC conditions. Because theBEH particles are more rugged than silica based HILIC phases, the BEHparticles provide chemical stability and further result in long columnlifetimes.

C. Mobile Phase

Mobile phase typically carries analytes through or across achromatographic device, such as a column. During the chromatography,analytes can interact with the stationary phase of the chromatographicdevice. Mobile phase of the chromatography can be a gas, liquid and anyother fluid. Based on the separation mechanism, the mobile phase can beaqueous, ionic or a highly organic. In certain embodiments of theinvention, the mobile phase is a highly organic solvent. In otherembodiments, the mobile phase comprises acetonitrile and water. In otherparticular embodiments, the mobile phase further comprises a polarsolvent, for example, isopropanol, n-propanol, methanol, ethanol andbutanol.

Typically the mobile phase for HILIC is mixture of water and a highorganic solvent. During the chromatography, the polar analytes, such asa glycopeptide or debris of glycoprotein in the invention, are retainedby the polar stationary phase. The dynamics between aqueous layer aroundthe stationary phase and the organic mobile phase normalize retention ofanalytes based on the polarity. Non-polar molecules, however, cannotbind or bind weakly to the chromatographic surface such that they can beeasily removed from the chromatography column by the mobile phase. Incertain embodiments, the mobile phase can include, without limitation,acetonitrile, isopropanol, n-propanol, methanol, ethanol, butanol, waterand a mixture thereof.

In another embodiment, an ion paring agent in the invention is ahydrophobic counterion and an additive to the mobile phase. Ion pairingagents mostly form ion pairing with positively charged residues of theprotein-related components found in glycoproteins/glycopeptides, but notwith glycans because glycans may not be cationic. Therefore, the proteinportion cannot extensively contribute to partitioning or binding to thestationary phase. As a consequence, glycan moieties predominatelyinteract with the stationary phase and the separation is determinedlargely by the glycan moiety. Thus, the glycoproteins/glycopeptidesmodified with different glycans can be separated. As evidence, FIG. 3shows the effect of ion pairing agents, and that ion pairing toprotonated positively charged protein residue simplifies the HILICretention mechanism and enhances selectivity for the glycans while theyare covalently attached to peptide/proteinaceous chains. Ion pairingagents can include, without limitation, trifluoroacetic acid (TFA),heptafluorobutyric acid (HFBA), pentafluoropropionic acid (PFPA),nonafluoropentanoic acid (NFPA), acetic acid, propanoic acid, orbutanoic acid. In particular embodiments, the use of TFA may bebeneficial, not only because it controls pH and ion pairing, but alsobecause it permits sensitive, low wavelength UV detection and isMS-compatible.

D. Operation

a. Column Pressure

According to the invention, the column inlet pressure for chromatographyoperation is elevated to minimize aggregation of the loaded sample,e.g., glycoproteins. In certain embodiments, the column inlet pressurecan be no less than 3000 psi, 4000 psi, 5000 psi, 6000 psi, or 7000 psi.

As shown in FIGS. 4A and 4B, protein aggregation on column is reduced oreliminated and the retention times of such sample analytes are reduced.

b. Aqueous Diluent and Volumes of Injection

In certain embodiments, an aqueous sample containingglycoprotein/glycopeptides is injected onto the HILIC column to increasethe ease of use of the invention. In certain embodiments, the samplecontaining glycoprotein is loaded onto a 2.1 mm ID column in a volumeless than 10.0 μL, less than 9.0 μL, less than 8.0 μL, less than 7.0 μL,less than 6.0 μL, less than 5.0 μL, less than 4.0 μL, less than 3.0 μL,less than 2.0 μL, or less than 1.5 μL.

In yet other embodiments, an aqueous sample containing glycoprotein isloaded such that an injection volume is less than 1/100^(th) of thechromatography column volume. Low volume injections of aqueous samplesonto a HILIC column is against common practice, in which sample diluentsare more often made to match initial mobile phase composition. Inanother embodiments, the sample injection volume can be any volume ifthe sample diluents have compatible dielectric strength of the initialmobile phase composition.

As shown in FIG. 5, a 2.1×150 mm column of this invention operated witha flow rate of 0.2 mL/min can tolerate an injection of aqueous sample upto 1.5 μL.

c. Operating Temperature

According to the invention, the operating temperature of thechromatography can be of between about 0° C. and 100° C., between about10° C. and 80° C., between about 30° C. and 80° C., between about 50° C.and 80° C., between about 60° C. and 80° C., or between about 70° C. and80° C. For example, FIG. 6 is showing comparative views of separation ofRNase B and intact trastuzumab at different column temperatures of theranges from 30° C. to 80° C.

d. Flow Rate

In certain embodiments of the invention, wherein a 2.1 mm ID column isemployed, the flow rate of the mobile phase can be between about 0.1mL/min and 1.0 mL/min, between about 0.1 mL/min and 0.9 mL/min, betweenabout 0.1 mL/min and 0.8 mL/min, between about 0.1 mL/min and 0.7mL/min, between about 0.1 mL/min and 0.6 mL/min, between about 0.1mL/min and 0.5 mL/min, between about 0.1 mL/min and 0.4 mL/min, orbetween about 0.2 mL/min and 0.3 mL/min. For example, FIG. 7 is achromatogram showing comparative view of separation of RNase B atdifferent flow rates, 0.1 mL/min, 0.2 mL/min, 0.3 mL/min, and 0.4mL/min.

Method for Characterizing a Glycoprotein or Glycopeptide Using HILICChromatography

In one embodiment of the invention, a method of performing hydrophilicchromatography (HILIC) for characterizing a glycopeptide comprisingsteps of

-   -   a) preparing a sample containing the glycopeptide in a sample        diluent;    -   b) providing a column having an inlet and an outlet and a        stationary phase material in the column wherein the stationary        phase material comprises a plurality of pores;    -   c) loading the sample on the stationary phase material at a        column inlet pressure of no less than about 3,000 psi and        flowing the sample with a mobile phase eluent through said        column;    -   d) separating the sample from the outlet into one or more        fractions; and    -   e) identifying the fractions.

In particular embodiments, the stationary phase material comprises atleast one hydrophilic monomer and a poly-amide bonded phase.

In one embodiment, the glycopeptide is derived from a glycoprotein or aglycosylated monoclonal antibody.

In other embodiments of the invention, the method of the preparationstep of a) can be, without limitation, reduction, enzymatic digestion,denaturation, fragmentation, chemical cleavage or a combination thereof.

In certain embodiments, the stationary phase material of step of b) isfully porous or superficially porous.

In certain embodiments, the average diameter of the pores is greaterthan or equal to about 200 Å, greater than or equal to about 250 Å,greater than or equal to about 300 Å, or greater than or equal to about450 Å;

In another certain embodiments, the stationary phase has an average porediameter small enough in diameter to completely restrict access ofglycoproteins/glycopeptides from the pore network of the particle,wherein the average diameter of the pores is from about 1 to about 50 Å,from about 5 to about 40 Å, or from about 10 to about 30 Å.

In certain aspect, the stationary phase is in one or more forms ofporous particles, e.g., beads, pellets, or any other form desirable foruse. The porous particles can have, e.g., a spherical shape, a regularshape or an irregular shape. In some embodiments, the particles arebeads having a diameter in the range from about 0.1 to about 500 μm,from about 1 to about 100 μm, or from about 1 to about 10 μm. In yetanother certain aspect, the stationary phase material further comprisesa porous monolith.

In certain embodiments of the invention, the mobile phase is a highlyorganic solvent. In other embodiments, the mobile phase comprisesacetonitrile and water. In other particular embodiments, the mobilephase further comprises a polar solvent, for example, isopropanol,n-propanol, methanol, ethanol and butanol.

In other embodiments, an aqueous sample containing glycoprotein isloaded such that an injection volume is less than 1/100 of the columnvolume. In another embodiments, the sample injection volume can be anyvolume if the sample diluents have compatible dielectric strength of theinitial mobile phase composition.

In another certain embodiments, the sample diluent comprises adenaturant. The denaturant can be, without limitation, guanidinehydrochloride (GuHCl).

In certain embodiments of the invention, the method further comprises astep of adding an ion pairing agent to the mobile phase. The ion pairingagent can include, without limitation, trifluoroacetic acid (TFA),heptafluorobutyric acid (HFBA), pentafluoropropionic acid (PFPA),nonafluoropentanoic acid, acetic acid, propanoic acid, or butanoic acid.

In certain embodiments, the method further comprises elevating a columnpressure. In yet certain embodiments, the column pressure is no lessthan 3,000 psi, no less than 4,000 psi, no less than 5,000 psi, no lessthan 6,000 psi, or no less than 7,000 psi.

In still other embodiments, the identification step is archived byultraviolet detection, electrospray ionization (ESI)-mass spectrometry(MS), evaporative light scattering, fluorescence, mass spectrometry,matrix assisted laser desorption (MALDI)-MS, MALDI MS/MS, ESI MS/MS,MS^(n), MS^(e), nuclear magnetic resonance, infrared analysis or acombination thereof. In certain embodiments, the identification can beachieved by comparison of mass spectrometry peaks with known compoundsin a computer database.

In one aspect of the invention, a method of analyzing a glycopeptide ina sample, comprising a step of contacting said sample with a stationaryphase wherein said stationary phase comprises at least one hydrophilicmonomer and a poly-amide bonded phase with a plurality of pores, whereinthe average pore diameter greater than or equal to about 200 Å, greaterthan or equal to about 250 Å, greater than or equal to about 300 Å, orgreater than or equal to about 450 Å.

Any glycopeptide can be analyzed according to this invention. Theglycopeptide can be, but not limited to, derived from a glycoprotein, ora glycosylated monoclonal antibody.

In certain aspect, the stationary phase material is a fully porous or asuperficially porous material.

In another certain aspect, the stationary phase material is in one ormore forms of porous particles, e.g., beads, pellets, or any other formdesirable for use. The porous particles can have, e.g., a sphericalshape, a regular shape or an irregular shape. In some embodiments, theparticles are beads having a diameter in the range from about 0.1 toabout 500 m, from about 1 to about 100 m, or from about 1 to about 10 m.In yet another certain aspect, the stationary phase material furthercomprises a porous monolith.

In yet another aspect of the invention, the method further comprises astep of preparing said sample. The step of preparing the sample, can be,without limitation, reduction, enzymatic digestion, denaturation,fragmentation, chemical cleavage or a combination thereof.

In still another aspect of the invention, the method further comprises astep of adding an ion pairing agent to the mobile phase eluent. The ionpairing agent can be trifluoroacetic acid, heptafluorobutyric acid,pentafluoropropionic acid, nonafluoropentanoic acid, acetic acid,propanoic acid, or butanoic acid.

In certain aspect, the method further comprises elevating a columnpressure. It is preferred that the column pressure is no less than about3,000 psi, no less than about 4,000 psi, no less than about 5,000 psi,no less than about 6,000 psi, or no less than about 7,000 psi.

In still another preferred aspect, the method comprises identificationstep of the glycosylation of the glycopeptide, which can be achieved byultraviolet detection, ESI-MS, evaporative light scattering,fluorescence, mass spectrometry, MALDI-MS, ESI-MS, MALDI MS/MS, ESIMS/MS, MS^(n), MS^(e), nuclear magnetic resonance, infrared analysis ora combination thereof. In certain embodiments, the identification can beachieved by comparison of mass spectrometry peaks with known compoundsin a computer database.

Method for Characterizing Glycosylation of a Monoclonal Antibody UsingHILIC Chromatography

The methods of the invention also provide a novel chromatographic methodto characterize the glycosylation of monoclonal antibody (mAb)therapeutics by means of intact protein, fragment, and peptide-levelHILIC-UV-MS analyses.

In certain embodiments, a monoclonal antibody is degraded or fragmentedwith an enzyme, such as IdeS (Immunoglobulin degrading enzyme fromStreptococcus pyogenes) while the fragmented mAb still contains glycans.In particular embodiments, it was possible to partially resolve thedifferentiated glycoforms of intact trastuzumab, such as the speciescontaining two GOF glycans from a species containing one G0F and one G1Fglycan. As evidenced in FIGS. 4A-B, global assessment of the intacttrastuzumab glycan profile is possible with the stationary phasematerial in the invention. The intact trastuzumab applied to the HILICstationary phase of the invention may also be prepared and fragmentedinto Fc and Fd with IdeS. As evidenced in FIGS. 8A-B and FIG. 15,glycosylation of IdeS-fragmented trastuzumab or cetuximab have beenwell-characterized with the stationary phase material in the invention.As evidenced in FIG. 14, these methods are suitable for detectingdifferences in the glycan profiles/compositions of two different batchesof trastuzumab.

In another certain embodiments, the monoclonal antibody is subjectedprior to HILIC chromatography with a protease, such as Lys-C or trypsin.As consequence, these methods have been applied to obtain highresolution Lys-C (FIG. 9) and tryptic glycopeptide mapping of cetuximab(FIG. 16). As evidenced in FIG. 13, these inventions provide a methodfor obtaining comparable results and similar sorts of informationotherwise obtained by released glycan analysis techniques.

In another embodiments, the invention may also provide a method toresolve protein modifications other than glycosylation at the intactprotein, fragment and peptide-levels of analysis. For example, thisinvention may be of use for chromatographically resolving a profile ofan antibody drug conjugate, wherein antibody species containingdifferent numbers of conjugates are separated.

Uses and Applications

The novel materials of the invention, e.g., in the form of porousparticles or monoliths, can be used for in any traditional form ofseparation. In particular, the novel materials of the invention, e.g.,in the form of porous particles or monoliths, can be used forhydrophilic interaction chromatography. Thus, the invention alsoprovides a porous material for hydrophilic interaction chromatography orchromatography comprising at least one ion-exchange functional group, atleast one hydrophilic component and at least one hydrophobic component.The ion-exchange functional groups enable the porous material tointeract with anionic, cationic, acidic and/or basic solutes. Thehydrophilic polar components enable the porous material to have polarinteractions and hydrogen bonding capabilities with solutes. Thehydrophobic components enable the porous material to have affinitytowards nonpolar solutes through hydrophobic interaction. Since theporous materials of this invention have a combination of variousinteraction forces towards solutes, they are very useful materials for,e.g., hydrophilic interaction chromatography, ion-exchange, and liquidchromatography applications. For example, these novel porous materialscan be used to bind, recover and/or remove solutes from fluids.Similarly, these novel porous materials have certain chemical affinitiesor attractions between the materials and certain molecules, particularlybiological or biochemical molecules, such as proteins, peptides,hormones, oligonucleotides, polynucleotides, vitamins, cofactors,metabolites, lipids and carbohydrates. As such, the materials of theinvention may be used to selectively adsorb and isolate certainbiomolecules for analysis and or quantification.

The invention also provides a method for removing or isolating acomponent, e.g., a solute, from a mixture. A solution having a solute iscontacted with a porous material of the invention under conditions thatallow for sorption of the solute to the porous material.

The solute can be, e.g., any molecule having a hydrophobic, hydrophilic,or ionic interaction or a combination of two or three of theseinteractions. Preferably, the solute is an organic compound of polaritysuitable for adsorption onto the porous material. Such solutes include,e.g., drugs, pesticides, herbicides, toxins and environmentalpollutants, e.g., resulting from the combustion of fossil fuels or otherindustrial activity, such as metal-organic compounds comprising a heavymetal such mercury, lead or cadmium. The solutes can also be metabolitesor degradation products of the foregoing materials. Solutes alsoinclude, e.g., biomolecules, such as proteins, peptides, hormones,oligonucleotides, polynucleotides, vitamins, cofactors, metabolites,lipids and carbohydrates. Solutes also include, e.g., modified proteins,modified oligonucleotides, single-stranded oligonucleotides,double-stranded oligonucleotides, DNA, and RNA.

In certain embodiments, the materials of the invention can be used forthe separation of intact proteins, polymeric biomolecules, biomoleculederived products, biomimetics, dendric, synthetic, and non-naturallyoccurring biomolecules, and the like

The solution e.g., can comprise water, an aqueous solution, or a mixtureof water or an aqueous solution and a water-miscible polar organicsolvent, e.g., methanol, ethanol, N, N-dimethylformamide,dimethylsulfoxide or acetonitrile. In a preferred embodiment, thesolution is an acidic, basic or neutral aqueous, i.e., between about 0%and about 99% water by volume, solution. Specific examples are providedin the experimentals. The solution comprising the solute can,optionally, further contain one or more additional solutes. In oneembodiment, the solution is an aqueous solution which includes a complexvariety of solutes. Solutions of this type include, e.g., cell cultureextracts, blood, plasma, urine, cerebrospinal fluid, synovial fluid andother biological fluids, including, e.g., extracts of tissues, such asliver tissue, muscle tissue, brain tissue or heart tissue. Such extractscan be, e.g., aqueous extracts or organic extracts which have been driedand subsequently reconstituted in water or in a water/organic mixture.Solutions also include, e.g., ground water, surface water, drinkingwater or an aqueous or organic extract of an environmental sample, suchas a soil sample. Other examples of solutions include a food substance,such as a fruit or vegetable juice or milk or an aqueous oraqueous/organic extract of a food substance, such as fruit, vegetable,cereal or meat. Other solutions include, e.g., natural productsextractions from plants and broths.

The solution can be contacted with the porous material in any fashionwhich allows sorption of the solute to the porous material, such as abatch or chromatographic process. For example, the solution can beforced through a porous polymer column, disk or plug, or the solutioncan be stirred with the porous material, such as in a batch-stirredreactor. The solution can also be added to a porous material-containingwell of a microtiter plate. The porous material can take the form of amonolith or particle, e.g., beads or pellets. The solution is contactedwith the porous material for a time period sufficient for the solute ofinterest to substantially sorb onto the porous material. This period istypically the time necessary for the solute to equilibrate between theporous material surface and the solution. The sorption or partition ofthe solute onto the porous material can be partial or complete.

The invention also includes a method for analytically determining thelevel of solute in a solution. A solution having a solute is contactedwith a porous material under conditions so as to allow sorption of thesolute to the porous material. The material comprises at least oneion-exchange functional group, at least one hydrophilic polar componentand at least one hydrophobic component. The porous material having thesorbed solute is washed with a solvent under conditions so as to desorbthe solute from the porous material. The level of the desorbed solutepresent in the solvent after the washing is analytically determined.

The solution contacted with the porous material can comprise the soluteof interest in dilute form, e.g., at a concentration too low foraccurate quantitation. By sorbing the solute onto the porous materialand then, e.g., desorbing the solute with a substantially smaller volumeof a less polar solvent, a solution which includes the solute ofinterest can be prepared having a substantially higher concentration ofthe solute of interest than that of the original solution. The methodcan also result in solvent exchange, that is, the solute is removed froma first solvent and re-dissolved in a second solvent.

Solvents which are suitable for desorbing the solute from the porousmaterial can be, e.g., polar water-miscible organic solvents, such asalcohols, e.g., methanol, ethanol or isopropanol, acetonitrile, acetone,and tetrahydrofuran, or mixtures of water and these solvents. Thedesorbing solvent can also be, e.g., a nonpolar or moderately polarwater-immiscible solvent such as dichloromethane, diethylether,chloroform, or ethylacetate. Mixtures of these solvents are alsosuitable. Preferred solvents or solvent mixtures must be determined foreach individual case. Specific examples are provided with theexperimental details. A suitable solvent can be determined by one ofordinary skill in the art without undue experimentation, as is routinelydone in chromatographic methods development (see, e.g., “A Resource forSample Preparation Methods Development,” 6th edition, Waters, Milford,Mass. (1995); Snyder and Kirkland, Introduction to Modern LiquidChromatography, New York: J. Wiley and Sons (1974)).

The level of the desorbed solute present in the solvent can beanalytically determined by a variety of techniques known to thoseskilled in the art, e.g., high performance liquid chromatography, liquidchromatography/mass spectrometry, gas chromatography, gaschromatography/mass spectrometry, or immunoassay.

The invention also provides separation devices comprising the porousmaterials of the invention. Such devices include chromatographiccolumns, cartridges, thin layer chromatographic plates, filtrationmembranes, sample clean up devices, solid phase organic synthesissupports, and microtiter plates. In certain embodiments, more than onetype of functionalized porous material can be used in the separationdevices, e.g., columns, cartridges, and the like.

As noted above, the porous materials of the invention are especiallywell suited for hydrophilic interaction chromatography. Thus, theinvention also includes a hydrophilic interaction chromatographycartridge comprising a porous material of the invention packed inside anopen-ended container. In one embodiment, the porous material is packedas particles within the open-ended container to form a hydrophilicinteraction chromatography cartridge.

The container can be, e.g., a cylindrical container or column, which isopen at both ends so that the solution can enter the container throughone end, contact the porous material within the container, and exit thecontainer through the other end. In the form of porous particles, theporous material can be packed within the container as small particles,such as beads having a diameter between about 0.1 μm and about 500 μm;between about 1 μm and about 100 μm; or between about 1 μm and about 10μm. In certain embodiments, the porous particles can be packed in thecontainer enmeshed in a porous membrane.

The container can be formed of any material, which is compatible, withinthe time frame of the hydrophilic interaction chromatography process,with the solutions and solvents to be used in the procedure. Suchmaterials include glass, various plastics, such as high densitypolyethylene and polypropylene, and various metals, such as steel andtitanium. In one embodiment, the container is cylindrical through mostof its length and has a narrow tip at one end. One example of such acontainer is a syringe barrel. The amount of porous material within thecontainer is limited by the container volume and can range from about0.001 g to about 50 kg, and preferably is between about 0.025 g andabout 1 g. The amount of porous material suitable for a given extractiondepends upon the amount of solute to be sorbed, the available surfacearea of the porous material and the strength of the interaction betweenthe solute and the porous material. This amount can be readilydetermined by one of ordinary skill in the art. The cartridge can be asingle use cartridge, which is used for the treatment of a single sampleand then discarded, or it can be used to treat multiple samples.

EXAMPLES

Materials.

All materials were used as received, except as noted. N-vinylcaprolactamand NVP were obtained from ISP, Sodium oleyl sulfate was obtained fromALCOLAC. Diethethylbenzene, 2-ethylhexanol, were obtained from ALDRICH.AIBN was obtained from DUPONT. Methocel E-15 and Divinylbenzene werepurchased from DOW. Inhibitor was removed from DVB prior to use.

Characterization

Those skilled in the art will recognize that equivalents of thefollowing instruments and suppliers exist and, as such, the instrumentslisted below are not to be construed as limiting.

The % C values were measured by combustion analysis (CE-440 ElementalAnalyzer; Exeter Analytical Inc., North Chelmsford, Mass.) or byCoulometric Carbon Analyzer (modules CM5300, CM5014, UIC Inc., Joliet,Ill.). The specific surface areas (SSA), specific pore volumes (SPV) andthe average pore diameters (APD) of these materials were measured usingthe multi-point N₂ sorption method (Micromeritics ASAP 2400;Micromeritics Instruments Inc., Norcross, Ga.). The SSA was calculatedusing the BET method, the SPV was the single point value determined forP/P₀>0.98 and the APD was calculated from the desorption leg of theisotherm using the BJH method. The micropore surface area (MSA) wasdetermined as the cumulative adsorption pore diameter data for pores <34Å subtracted from the specific surface area (SSA). The median mesoporediameter (MPD) and mesopore pore volume (MPV) were measured by mercuryporosimetry (Micromeritics AutoPore IV, Micromeritics, Norcross, Ga.).Skeletal densities were measured using a Micromeritics AccuPyc 1330Helium Pycnometer (V2.04N, Norcross, Ga.). Scanning electron microscopic(SEM) image analyses were performed (JEOL JSM-5600 instrument, Tokyo,Japan) at 7 kV. High resolution SEM image analyses were performed usinga Focused Ion Beam (FIB/SEM) instrument (Helios 600 Nanolab, FEICompany, Hillsboro, Oreg.) at 20 kV. Particle sizes were measured usinga Beckman Coulter Multisizer 3 analyzer (30-μm aperture, 70,000 counts;Miami, Fla.). The particle diameter (dp) was measured as the 50%cumulative diameter of the volume based particle size distribution. Thewidth of the distribution was measured as the 90% cumulative volumediameter divided by the 10% cumulative volume diameter (denoted 90/10ratio). Viscosity was determined for these materials using a Brookfielddigital viscometer Model DV-II (Middleboro, Mass.). FT-IR spectra wereobtained using a Bruker Optics Tensor 27 (Ettlingen, Germany).Multinuclear (¹³C, ²⁹Si) CP-MAS NMR spectra were obtained using a BrukerInstruments Avance-300 spectrometer (7 mm double broadband probe). Thespinning speed was typically 5.0-6.5 kHz, recycle delay was 5 sec. andthe cross-polarization contact time was 6 msec. Reported ¹³C and ²⁹SiCP-MAS NMR spectral shifts were recorded relative to tetramethylsilaneusing the external standards adamantane (¹³C CP-MAS NMR, δ −38.55) andhexamethylcyclotrisiloxane (²⁹Si CP-MAS NMR, 8-9.62). Populations ofdifferent silicon environments were evaluated by spectral deconvolutionusing DMFit software. [Massiot, D.; Fayon, F.; Capron, M.; King, I.; LeCalvé, S.; Alonso, B.; Durand, J.-O.; Bujoli, B.; Gan, Z.; Hoatson, G.Magn. Reson. Chem. 2002, 40, 70-76]

Example 1

Porous ethylene-bridged hybrid particles (60 g, 1.80 μm, SSA=88 m²/g;SPV=0.66 cm³/g; APD=300 Å; 6.41% C), prepared following the methoddescribed in U.S. Pat. No. 6,686,035, were surface modified using amodified process as detailed in U.S. Pat. No. 4,835,058 A, Example 6, inwhich 3-methacryloxypropyltrimethoxysilane was replaced with3-methacryloxypropyltrichlorosilane. In this process themethacryloxypropyl surface modified particles were exposed to anacetone/ammonium acetate solution similar to that detailed in U.S. Pat.No. 6,686,035, example 24. The product of this reaction had 7.65% C.

Example 2

The material of Example 1 was further reacted with acrylamide andpotassium persulfate in a methanol solution as detailed in U.S. Pat. No.4,835,058 A, Example 6. The product is dried in a vacuum oven at 30-50°C. The product of this reaction had 1.12-1.20% N.

Example 3

The process of Example 1 and 2 is modified replacing3-methacryloxypropyltrichlorosilane with one or more of the following;3-methacryloxypropylmethyldichlorosilane,3-methacryloxypropyldimethylchlorosilane,3-methacryloxypropyltrimethoxysilane,3-methacryloxypropylmethyldimethoxysilane,3-methacryloxypropyldimethylmethoxysilane,3-methacryloxypropyltriethoxysilane,3-methacryloxypropylmethyldiethoxysilane,3-methacryloxypropyldimethylethoxysilane,3-acryloxypropyltrichlorosilane, 3-acryloxypropylmethyldichlorosilane,3-acryloxypropyldimethylchlorosilane 3-acryloxypropyltrimethoxysilane,3-acryloxypropylmethyldimethoxysilane,3-acryloxypropyldimethylmethoxysilane, 3-acryloxypropyltriethoxysilane,3-acryloxypropylmethyldiethoxysilane,3-acryloxypropyldimethylethoxysilane, styrylethyltrichlorosilane,styrylethylmethyldichlorosilane, styrylethyldimethylchlorosilane,styrylethyltrimethoxysilane, styrylethylmethyldimethoxysilane,styrylethyldimethylmethoxysilane, styrylethyltriethoxysilane,styrylethylmethyldiethoxysilane, styrylethyldimethylethoxysilane,vinyltriethoxysilane, vinyltrimethoxysilane,N-(3-acryloxy-2-hydroxypropyl)-3-aminopropyl triethoxysilane,(3-acryloxypropyl) trimethoxysilane,O-(methacryloxyethyl)-N-(triethoxysilylpropyl)urethane,N-(3-methacryloxy-2-hydroxypropyl)-3-aminopropyl triethoxysilane,methacryloxy methyltriethoxysilane, methacryloxymethyl trimethoxysilane,methacryloxypropy methyldiethoxysilane, methacryloxypropylmethyldimethoxysilane, methacryl oxypropyltris (methoxyethoxy)silane,3-(N-styrylmethyl-2-aminoethylamino) propyltrimethoxysilanehydrochloride,

Example 4

The process of Example 2-3 is modified replacing acrylamide with one ormore of the following; divinylbenzene, styrene, ethylene glycoldimethacrylate, 1-vinyl-2-pyrrolidinone and tert-butylmethacrylate,acrylamide, methacrylamide, N,N′-(1,2-dihydroxyethylene)bisacrylamide,N,N′-ethylenebisacrylamide, N,N′-methylenebisacrylamide, butyl acrylate,ethyl acrylate, methyl acrylate, 2-(acryloxy)-2-hydroxypropylmethacrylate, N,N-bis(2-cyanoethyl)acrylamide,N-acryloyltris(hydroxymethyl)aminomethane, 3-(acryloxy)-2-hydroxypropylmethacrylate, trimethylolpropane triacrylate, trimethylolpropaneethoxylate triacrylate, tris[(2-acryloyloxy)ethyl] isocyanurate,acrylonitrile, methacrylonitrile, itaconic acid, methacrylic acid,trimethylsilylmethacrylate, N-[tris(hydroxymethyl)methyl]acrylamide,(3-acrylamidopropyl)trimethylammonium chloride,[3-(methacryloylamino)propyl]dimethyl(3-sulfopropyl)ammonium hydroxideinner salt,

and the monomers detailed in Peter A. G. Cormack, Journal ofChromatography B, 804 (2004) 173-182 are included herein in theirentirety.

Example 5

The process of Example 2-4 is modified wherein the monomers used in thepolymerization are binary mixture of 35 to 99 mole percent acrylamidewith 65 to 1 mole percent of one of the following following;N,N-methylenebisacrylamide, N,N-ethylenebisacrylamide,N,N-propylenebisacrylamide, N,N-butylenebisacrylamide,N,N′-(1,2-dihydroxyethylene)bisacrylamide, and1,4-bis(acryloyl)piperazine. Materials prepared by this approach haveincreased crosslinking in the bonded phase. This improves the chemicalstability when exposed to acid and basic mobile phases. It also improvesthe temperature stability, allows for reduced chemical bleed (asobserved by UV or MS detection), and allows for improved pressurestability when columns are used in gradient mode or have changes inmobile phase composition (e.g., different ratios of acetonitrile towater).

Example 6

The process of Examples 2-5 is modified to use a binary, ternary orgreater mixture of monomers detailed in Example 4 and 5.

Example 7

The process of Examples 2-6 is modified to replace potassium persulfatewith one of the following; tert-amyl peroxybenzoate,4,4-azobis(4-cyanovaleric acid), 1,1′-azobis(cyclohexanecarbonitrile),2,2′-azobisisobutyronitrile, benzoyl peroxide,2,2-bis(tert-butylperoxy)butane, 1,1-bis(tert-butylperoxy)cyclohexane,2,5-bis(tert-butylperoxy)-2,5-dimethylhexane,2,5-bis(tert-butylperoxy)-2,5-dimethyl-3-hexyne,bis(1-(tert-butylperoxy)-1-methylethyl)benzene,1,1-bis(tert-butylperoxy)-3,3,5-trimethylcyclohexane, tert-butylhydroperoxide, tert-butyl peracetate, tert-butyl peroxide, tert-butylperoxybenzoate, tert-butylperoxy isopropyl carbonate, cumenehydroperoxide, cyclohexanone peroxide, dicumyl peroxide, lauroylperoxide, 1,2,4-pentanedione peroxide, and peracetic acid.

Example 8

The process of Examples 1-7 is modified to use a silane that has aterminal group that can be used with Reversible Addition FragmentationChain Transfer (RAFT) of any of the monomers detailed in Examples 4-6.Included in this are use of silanes of the formula

A(CR⁸R⁹)_(n)Si(Y)_(3-x)(R′)_(x)  (equation 1)

where n=1-30, advantageously 2-3;x is 0-3; advantageously 0;Y represents chlorine, dimethylamino, triflate, methoxy, ethoxy, or alonger chain alkoxy group;R⁸ and R⁹ are independently hydrogen, methyl, n-alkyl, branched alkyl,aryl, napthyl, heterocycle, carboxylic acid, or cyanoR′ independently represents an alkyl, branched alkyl, aryl, orcycloalkyl group;A represent a group that is utilized in RAFT polymerizations, including(but not limited to):

Where each example of R¹⁰, R¹¹, R¹² and R¹³ are independently hydrogen,methyl, n-alkyl, branched alkyl, aryl, napthyl, heterocycle, carboxylicacid, or cyano;n¹⁰, n¹², and n¹³ are 0-30;X¹⁰, when present, is a carbamate, carbonate, thioether, ether, amide,urea, epoxide, polyethylene glycol, ethylene glycol, sugar, alkyl, aryl,or heterocycle group.n¹¹ is 0 or 1;Conditions and additional reagents used for RAFT are detailed inHandbook of RAFT Polymerization, Christopher Barner-Kowollik (Wiley-VCH:Weinheim, Germany), 2008, and references therein. This reference isincluded herein in its entirety.

Example 9

The process of Examples 1-7 is modified to use a silane that has aterminal group that can be used with Atom Transfer RadicalPolymerization (ATRP) of any of the monomers detailed in Examples 4-6.Included in this area use of silanes of the formula

X(R⁸R⁹)_(n)Si(Y)_(3-x)(R′)_(x)  (equation 2)

where n=1-30, advantageously 2-3;x is 0-3; advantageously 0;Y represents chlorine, dimethylamino, triflate, methoxy, ethoxy, or alonger chain alkoxy group;R⁸ and R⁹ are independently hydrogen, methyl, n-alkyl, branched alkyl,aryl, napthyl, heterocycle, carboxylic acid, or cyanoR′ independently represents an alkyl, branched alkyl, aryl, orcycloalkyl group;X represent a group that is utilized in ATRP, including (but not limitedto):

Where each example of R¹² and R¹³ are independently hydrogen, methyl,n-alkyl, branched alkyl, aryl, napthyl, heterocycle, cycloalkyl,carboxylic acid, or cyano;n¹⁴ is 0-30;X¹¹, when present, is a carbamate, carbonate, thio-ether, ether, amide,urea, epoxide, polyethylene glycol, ethylene glycol, sugar, alkyl, aryl,or heterocycle group.n¹¹ is 0 or 1;Conditions and additional reagents used for ATRP are detailed in Silanesand Other Coupling Agents, Volume 5, K. L. Mittal (VSP/Brill: Leiden,The Netherlands), 2009, and references therein. This reference isincluded herein in its entirety.

Example 10

The process of Examples 2-9 is modified to use different solvent systemsduring the polymerization step. Solvents for these reactions include(but are not limited to) one or more of the following: methanol,ethanol, water, propanol, toluene, benzene, zylene, trimethylbenzene,paraffin, dimethyl formamide, dimethylsulfoxane, dioxane, ethyleneglycol, dimethyl ethylene glycol, hexanes, methylene chloride,chloroform, and supercritical carbon dioxide.

Example 11

The process of Examples 1-10 is modified to use an additional surfacemodification following the general procedure shown in Example 1;including one or more of the following: aminopropyltriethoxysilane,aminopropyltrimethoxysilane, 2-(2-(trichlorosilyl)ethyl)pyridine,2-(2-(trimethoxy)ethyl)pyridine, 2-(2-(triethoxy)ethyl)pyridine,2-(4-pyridylethyl)triethoxysilane, 2-(4-pyridylethyl)trimethoxysilane,2-(4-pyridylethyl)trichlorosilane, chloropropyltrimethoxysilane,chloropropyltrichlorosilane, chloropropyltrichlorosilane,chloropropyltriethoxysilane, imidazolylpropyltrimethoxysilane,imidazolylpropyltriethoxysilane, imidazolylpropyl trichlorosilane,sulfopropyltrisilanol, carboxyethylsilanetriol,2-(carbomethoxy)ethylmethyldichlorosilane,2-(carbomethoxy)ethyltrichlorosilane,2-(carbomethoxy)ethyltrimethoxysilane,n-(trimethoxysilylpropyl)ethylenediamine triacetic acid,(2-diethylphosphatoethyl)triethoxysilane,3-mercaptopropyltriethoxysilane, 3-mercaptopropyltrimethoxysilane,bis[3-(triethoxysilyl)propyl]disulfide,bis[3-(triethoxysilyl)propyl]tetrasulfide,2,2-dimethoxy-1-thia-2-silacyclopentane,bis(trichlorosilylethyl)phenylsulfonyl chloride,2-(chlorosulfonylphenyl)ethyltrichlorosilane,2-(chlorosulfonylphenyl)ethyltrimethoxysilane,2-(ethoxysulfonylphenyl)ethyltrimethoxysilane,2-(ethoxysulfonylphenyl)ethyltrimethoxysilane,2-(ethoxysulfonylphenyl)ethyltrichlorosilane, sulphonic acidphenethyltrisilanol, (triethoxysilyl ethyl)phenyl phosphonic aciddiethyl ester, (trimethoxysilyl ethyl)phenyl phosphonic acid diethylester, (trichlorosilyl ethyl)phenyl phosphonic acid diethyl ester,phosphonic acid phenethyltrisilanol, N-(3-trimethoxysilylpropyl)pyrrole,N-(3-triethoxysilylpropyl)-4,5-dihydroimidazole,bis(methyldimethoxysilylpropyl)-N-methylamine,tris(triethoxysilylpropyl)amine,bis(3-trimethoxysilylpropyl)-N-methylamine,(N,N-diethyl-3-aminopropyl)trimethoxysilane,N-(hydroxyethyl)-N-methylaminopropyltrimethoxysilane,3-(N,N-dimethylaminopropyl)trimethoxysilane,bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane,N,N′-bis(hydroxyethyl)-N,N′-bis(trimethoxysilylpropyl)ethylenediamine,or N,N-dimethyl-3-aminopropylmethyldimethoxysilane.

Example 12

The process of Examples 1-11 is modified to use an additional surfacemodification following the general procedure shown in Example 1;including one or more of the following:

Z_(a)(R′)_(b)Si—R″, where Z═Cl, Br, I, C₁-C₅ alkoxy, dialkylamino ortrifluoromethanesulfonate; a and b are each an integer from 0 to 3provided that a+b=3; R′ is a C1-C6 straight, cyclic or branched alkylgroup, and R″ is a functionalizing group.

In another embodiment, the materials are surface modified by coatingwith a polymer.

In certain embodiments, R′ is selected from the group consisting ofmethyl, ethyl, propyl, isopropyl, butyl, t-butyl, sec-butyl, pentyl,isopentyl, hexyl and cyclohexyl. In other embodiments, R′ is selectedfrom the group consisting of alkyl, alkenyl, alkynyl, aryl, cyano,amino, diol, nitro, ester, a cation or anion exchange group, an alkyl oraryl group containing an embedded polar functionality and a chiralmoiety. In certain embodiments, R′ is selected from the group consistingof aromatic, phenylalkyl, fluoroaromatic, phenylhexyl,pentafluorophenylalkyl and chiral moieties.

In one embodiment, R″ is a C₁-C₃₀ alkyl group. In a further embodiment,R″ comprises a chiral moiety. In another further embodiment, R″ is aC₁-C₂₀ alkyl group.

In certain embodiments, the surface modifier comprises an embedded polarfunctionality. In certain embodiments, such embedded polar functionalityincludes carbonate, amide, urea, ether, thioether, sulfinyl, sulfoxide,sulfonyl, thiourea, thiocarbonate, thiocarbamate, ethylene glycol,heterocyclic, or triazole functionalities. In other embodiments, suchembedded polar functionality includes carbamate functionalities such asdisclosed in U.S. Pat. No. 5,374,755, and chiral moieties. Such groupsinclude those of the general formula

wherein l, m, o, r and s are 0 or 1, n is 0, 1, 2 or 3 p is 0, 1, 2, 3or 4 and q is an integer from 0 to 19; R₃ is selected from the groupconsisting of hydrogen, alkyl, cyano and phenyl; and Z, R′, a and b aredefined as above. Preferably, the carbamate functionality has thegeneral structure indicated below:

wherein R⁵ may be, e.g., cyanoalkyl, t-butyl, butyl, octyl, dodecyl,tetradecyl, octadecyl, or benzyl. Advantageously, R⁵ is octyl, dodecyl,or octadecyl.

In certain embodiments, the surface modifier is selected from the groupconsisting of phenylhexyltrichlorosilane,pentafluorophenylpropyltrichlorosilane, octyltrichlorosilane,octadecyltrichlorosilane, octyldimethylchlorosilane andoctadecyldimethylchlorosilane. In some embodiments, the surface modifieris selected from the group consisting of octyltrichlorosilane andoctadecyltrichlorosilane. In other embodiments, the surface modifier isselected from the group consisting of an isocyanate or1,1′-carbonyldiimidazole (particularly when the hybrid group contains a(CH₂)₃OH group).

Example 13

The process of Examples 1-12 is modified to replace the BEH, 300 Åmaterials with different materials. Included in this are monolithic,spherical, granular, superficially porous and irregular materials thatare silica, hybrid organic/inorganic materials, hybrid inorganic/organicsurface layers on hybrid inorganic/organic, silica, titania, alumina,zirconia, polymeric or carbon materials, and silica surface layers onhybrid inorganic/organic, silica, titania, alumina, zirconia orpolymeric or carbon materials. The particle size for spherical, granularor irregular materials vary from 0.5-100 μm; more preferably 1-50 μm;more preferably 1.5-10 μm; more preferably 1.7-5 μm. The APD for thesematerials vary from 200 to 2,000 Å; more preferably 250 to 1,000 Å; morepreferably 300 to 1,000 Å. The SSA for these materials vary from 20 to1000 m²/g; more preferably 90 to 800 m²/g; more preferably 150 to 600m²/g; more preferably 300 to 550 m²/g. The TPV for these materials varyfrom 0.2 to 1.5 cm³/g; more preferably 0.5 to 1.2 cm³/g; more preferably0.7 to 1.1 cm³/g. The macropore diameter for monolithic materials varyfrom 0.1 to 30 m, more preferably 0.5 to 25 μm, more preferably 1 to 20μm. For these materials the polymerization step is performed in a mannerto fill 5-100% of the particle particle porosity; more preferably 10-90%of the particle porosity; more preferably 20-80% of the particleporosity; more preferably 30-70% of the particle porosity; morepreferably 40-60% of the particle porosity as measured by multi-point N₂sorption method or by mercury porosimetry. The nitrogen content of theproduct is increased in this process.

Example 14

The process of Examples 1-12 is modified to replace the BEH, 300 Åmaterials with spherical BEH material having 450 Å average porediameter. For these materials the polymerization step is performed in amanner to fill 5-100% of the particle porosity; more preferably 10-90%of the particle porosity; more preferably 20-80% of the particleporosity; more preferably 30-70% of the particle porosity; morepreferably 40-60% of the particle porosity as measured by multi-point N₂sorption method or by mercury porosimetry. The nitrogen content of theproduct is increased in this process.

Example 15

The process of Examples 1-12 is modified to replace the BEH, 300 Åmaterials with spherical BEH material having 900-1,000 Å average porediameter. For these materials the polymerization step is performed in amanner to fill 5-100% of the particle porosity; more preferably 10-90%of the particle porosity; more preferably 20-80% of the particleporosity; more preferably 30-70% of the particle porosity; morepreferably 40-60% of the particle porosity as measured by multi-point N₂sorption method or by mercury porosimetry. The nitrogen content of theproduct is increased in this process.

Example 16

The process of Examples 1-12 is modified to replace the BEH, 300 Åmaterials with spherical nonporous silica. The particles size for thissilica vary from 0.5-100 μm; more preferably 1-50 μm; more preferably1.5-10 μm; more preferably 1.7-5 μm. The SSA for these materials varyfrom 0.5-500 m²/g; more preferably 1 to 100 m²/g; more preferably 2 to20 m²/g. The TPV for these materials is 0 to 0.1 cm³/g; more preferablyless than 0.08 cm³/g.

For these materials the polymerization step is performed in a manner toincrease the nitrogen content of the product in this process.

Example 17

The process of Examples 1-12 is modified to replace the BEH, 300 Åmaterials with spherical nonporous hybrid organic/inorganic particles.The particles size for this silica vary from 0.5-100 am; more preferably1-50 am; more preferably 1.5-10 am; more preferably 1.7-5 μm. The SSAfor these materials vary from 0.5-500 m²/g; more preferably 1 to 100m²/g; more preferably 2 to 20 m²/g. The TPV for these materials is 0 to0.1 cm³/g; more preferably less than 0.08 cm³/g. For these materials thepolymerization step is performed in a manner to increase the nitrogencontent of the product in this process.

Example 18

The process of Examples 1-12 is modified to replace the BEH, 300 Åmaterials with a wide-pore superficially porous silica particle,wide-pore superficially porous hybrid organic/inorganic particles,wide-pore superficially porous silica monoliths, and wide-poresuperficially porous hybrid organic/inorganic monoliths. The particlessize for this silica vary from 0.5-100 am; more preferably 1-50 am; morepreferably 1.5-10 am; more preferably 1.7-5 m. The APD for thesematerials vary from 200 to 2,000 Å; more preferably 250 to 1,000 Å; morepreferably 300 to 1,000 Å. The SSA for these materials vary from 10 to500 m²/g; more preferably 20 to 200 m²/g; more preferably 30 to 100m²/g. The TPV for these materials vary from 0.1 to 0.7 cm³/g; morepreferably 0.15 to 0.35 cm³/g; more preferably 0.2 to 0.3 cm³/g. Theratio of nonporous core diameter to final particle diameter (denotedRHO) vary from 0.5-0.99; more preferably 0.7-0.98; more preferably0.8-0.97; more preferably 0.85-0.96; more preferably 0.87-0.95; morepreferably 0.89-0.94; more preferably 0.90-0.93. The macropore diametersfor monolithic materials vary from 0.1 to 30 m, more preferably 0.5 tom, more preferably 1 to 20 m. For these materials the polymerizationstep is performed in a manner to fill 5-100% of the particle porosity;more preferably 10-90% of the particle porosity; more preferably 20-80%of the particle porosity; more preferably 30-70% of the particleporosity; more preferably 40-60% of the particle porosity as measured bymulti-point N₂ sorption method or by mercury porosimetry. The nitrogencontent of the product is increased in this process.

Example 19

The process of Examples 1-12 is modified to replace the BEH, 300 Åmaterials with a fully porous or superficially porous silica particle,hybrid organic/inorganic particle, silica monolith, or hybridorganic/inorganic monoliths with average pore diameters varying from 1to 50 Å; more preferably 5 to 40 Å; more preferably 10 to 30 Å. Theparticles size for this silica vary from 0.5-100 μm; more preferably1-50 μm; more preferably 1.5-10 μm; more preferably 1.7-5 μm. The SSAfor these materials vary from 10 to 500 m²/g; more preferably 20 to 200m²/g; more preferably 30 to 100 m²/g. The TPV for these materials varyfrom 0.1 to 0.7 cm³/g; more preferably 0.15 to 0.35 cm³/g; morepreferably 0.2 to 0.3 cm³/g. The ratio of nonporous core diameter tofinal particle diameter (denoted RHO) vary from 0.5-0.99; morepreferably 0.7-0.98; more preferably 0.8-0.97; more preferably0.85-0.96; more preferably 0.87-0.95; more preferably 0.89-0.94; morepreferably 0.90-0.93. The macropore diameters for monolithic materialsvary from 0.1 to 30 μm, more preferably 0.5 to 25 μm, more preferably 1to 20 μm. For these materials the polymerization step is performed in amanner to fill 5-100% of the particle porosity; more preferably 10-90%of the particle porosity; more preferably 20-80% of the particleporosity; more preferably 30-70% of the particle porosity; morepreferably 40-60% of the particle porosity as measured by multi-point N₂sorption method or by mercury porosimetry. The nitrogen content of theproduct is increased in this process.

Example 20

The process of Examples 1-19 is modified to use an additional surfacemodification following the general procedure shown in Example 1;including one or more of the following:

A combination of organic group and silanol group modification. In stillanother embodiment, the material has been surface modified by acombination of organic group modification and coating with a polymer. Ina further embodiment, the organic group comprises a chiral moiety. Inyet another embodiment, the material has been surface modified by acombination of silanol group modification and coating with a polymer. Inother embodiments, the material has been surface modified via formationof an organic covalent bond between the particle's organic group and themodifying reagent. In still other embodiments, the material has beensurface modified by a combination of organic group modification, silanolgroup modification and coating with a polymer. In another embodiment,the material has been surface modified by silanol group modification.

Example 21

The process of Examples 1-20 is modified to include additional resizing,washing, sedimentation, sonication and or treatment steps in one or moreof the following solvents: acetone, water, hexanes, toluene, methanol,ethanol, and supercritical carbon dioxide.

Example 22

The process of Examples 1-21 is modified using the following process:

a) one or more precursor materials detailed in Experiments 1, 13-19undergoes a primary surface modification using the process detailed inExamples 1, 3, 11 or 12, using the solvent systems detailed in Examples1, 3, 10-12;

b) These resulting material then undergoes a second surface modificationusing the process detailed in Examples 1, 3, 11 or 12, using the solventsystems detailed in Examples 1, 3, 10-12;

c) The resulting material then undergoes a third surface modificationusing the process detailed in Examples 2, 4-10;

d) The resulting material then undergoes additional processing asdetailed in Example 21.

Example 23

The process of Examples 1-22 is modified using the following process:

a) one or more precursor materials detailed in Experiments 1, 13-19undergoes a primary surface modification using the process detailed inExamples 1, 3, 11 or 12, using the solvent and conditions detailed inExamples 1, 3, 10-12;

b) These resulting material then undergoes a second surface modificationusing the process detailed in Examples 1, 3, 11 or 12, using the solventand conditions detailed in Examples 1, 3, 10-12;

c) The resulting material then undergoes a third surface modificationusing the process detailed in Examples 2, 4-10;

d) These resulting material then undergoes a second surface modificationusing the process detailed in Examples 1, 3, 11 or 12, using the solventand conditions detailed in Examples 1, 3, 10-12;

e) The resulting material then undergoes additional processing asdetailed in Example 21.

Example 24

The process of Examples 1-22 is modified using the following process:

a) one or more precursor materials detailed in Experiments 1, 13-19undergoes a primary surface modification using the process detailed inExamples 1, 3, 11 or 12, using the solvent and conditions detailed inExamples 1, 3, 10-12;

b) The resulting material then undergoes a second surface modificationusing the process detailed in Examples 2, 4-10;

d) These resulting material then undergoes a third surface modificationusing the process detailed in Examples 1, 3, 11 or 12, using the solventand conditions detailed in Examples 1, 3, 10-12;

e) The resulting material then undergoes additional processing asdetailed in Example 21.

Example 25

The process of Examples 22-23 is modified to have an initial surfacemodification with one or more silanes that have a polymerizable group asdetailed in Examples 1 or 3, followed by a second surface modificationwith one or more silanes that have an ionizable group from Example 11,followed by a third surface modification that further reacts thepolymerizable group, as detailed in Example 2, 4-10.

Example 26

The process of Examples 22-23 is modified to have an initial surfacemodification with one or more silanes that have a polymerizable group asdetailed in Examples 1 or 3, followed by a second surface modificationwith one or more silanes from Example 12, followed by a third surfacemodification that further reacts the polymerizable groups, as detailedin Example 2, 4-10.

Example 27

The process of Examples 22-23 is modified to have an three surfacemodifications performed sequentially in any order that comprise apolymerizable group as detailed in Examples 1 or 3; one or more silanesfrom Example 12, and one or more silanes that has an ionizable groupfrom Example 11. The resulting material further reacts the polymerizablegroup, as detailed in Example 2, 4-10.

Example 28

The process of Examples 27 is modified to have the three initial surfacemodifications performed concurrently in a one-pot reaction.

Example 29

The process of Examples 22-28 is modified to use one or more silanesthat has an ionizable group from Example 11; one or more polymerizablegroups as detailed in Examples 1 or 3; and further reacts thepolymerizable group, as detailed in Example 2, 4-10 using a one or morepolymerizable monomer that contain an ionizable groups.

Example 30

The process of Examples 29 is modified to have two or more ionizablegroups, at least one from a silane modification and at least one fromreacting a polymerizable monomer, in which all groups have the samecharge (e.g., acidic or basic groups).

Example 31

The process of Examples 29 is modified to have two or more ionizablegroups, at least one from a silane modification and at least one fromreacting a polymerizable monomer, in which groups have different charges(e.g., acidic, basic or zwitterionic groups).

Example 32

The process of Examples 22-31 wherein the resulting product contains azwitterionic group.

Example 33

The process of Examples 22-29 is modified to remove the additionalprocess steps detailed in Example 21.

Example 34

Superficially porous silica particles (1.9 μm, SSA=19 m²/g; APD=324 Å),were surface modified using the process detailed in Example 1 and 2. Theratio of nonporous core diameter to final particle diameter (denotedRho) of the feed particles was 0.84, determined by SEM and FIB-SEM. Theproduct of this reaction had 1.0% C.

Example 35

Superficially porous silica particles (1.7 μm, SSA=7 m²/g; APD=232 Å),were surface modified using the process detailed in Example 1 and 2. Theratio of nonporous core diameter to final particle diameter (denotedRho) of the feed particles was 0.93, determined by SEM and FIB-SEM. Theproduct of this reaction had 0.4% C.

Example 36

Porous silica particles (20 μm, SSA=326 m²/g; APD=104 Å), were surfacemodified using the process detailed in Example 1 and 2. The product ofthis reaction had 10.2% C and 2.07% N.

Example 37

Porous ethylene-bridged hybrid particles, prepared following the methoddescribed in U.S. Pat. No. 6,686,035, had a primary surface modificationusing 3-methacryloxypropyltrichlorosilane (MOS), as detailed in Example1, followed by a secondary reaction with acrylamide, as detailed inExample 2. Table 1 provides details on selected prototypes.

TABLE 1 Primary Surface Modification Precursor Nominal MOS SecondaryParticle Pore Surface Reaction with Size Diameter Coverage AcrylamideExample (μm) (Å) % C (μmol/m²) % N 37a 1.7 45 13.91 1.94 1.65 37b 1.7300 7.73 1.84 1.28 37c 1.7 300 7.66 1.80 1.14 37d 1.7 300 7.66 1.80 1.0737e 1.7 300 7.66 1.80 0.92 37f 1.7 300 7.65 2.00 1.12 37g 1.7 300 7.652.00 1.20 37h 1.7 300 7.77 1.70 1.22 37i 3.5 300 7.61 1.68 1.00 37j 3.5300 7.65 1.84 1.24

Example 38—Analysis of Glycoproteins

Materials/Reagents

LC/MS grade solvents (water and acetonitrile) were purchased from FisherScientific (Fair Lawn, N.J.) or Sigma-Aldrich (St. Louis, Mo.). Formicacid (FA) and trifluoroacetic acid (TFA) were obtained from Pierce(Rockford, Ill.). Ammonium acetate (Fluka 73594), ammonium formate(Fluka 70221), anthranilamide (2-AB, A89804), bovine ribonuclease B(R-7884), dithiothreitol (DTT), glu-fibrinopeptide b (F-3261),iodoacetamide, L-cysteine hydrochloride (Fluka 30119), proteomics-gradetrypsin (T6567), and Tris(2-carboxyethyl)phosphine (TCEP) were purchasedfrom Sigma-Aldrich (St. Louis, Mo.). Guanidine hydrochloride (GuHCl),hydroxylamine (NH₂OH) hydrochloride, iodoacetamide, sodium iodide, andurea were also from Sigma-Aldrich (St. Louis, Mo.). Monobasic sodiumphosphate was purchased from Acros Organics (New Jersey, U.S.A.).Achromobacter protease I (Lys-C) was purchased from Wako Co. (Richmond,Va.). Sodium hydroxide (10N) solution was obtained from J.T. Baker(Phillipsburg, N.J.). Concentrated hydrogen chloride was purchased fromFisher Scientific (Fair Lawn, N.J.). Trastuzumab (Herceptin, Genentech,South San Francisco, Calif.) and Cetuximab (Erbitux, ImClone Systems,Bridgewater, N.J.) were acquired from Besse Medical (West Chester,Ohio). Carboxypeptidase B was purchased from Worthington (Lakewood,N.J.). PNGase F was obtained from Prozyme (Hayward, Calif.). RapiGestsurfactant was acquired from Waters (Waters Corporation, Milford,Mass.). Immunoglobulin degrading enzyme from Streptoccocus pyogenes(IdeS, FabRICATOR) was obtained from Genovis (Lund, Sweden). Dimethylsulfoxide (DMSO), glacial acetic acid, and sodium cyanoborohydride wereacquired from Waters as part of a GlycoWorks Reagent Kit (186007034,Milford, Mass.).

HILIC Chromatography Optimization with a Model Glycosylated Protein,RNase B or Intact Trastuzumab

For general method development in the invention, the model RNase B isused. RNase B is used as intact glycoprotein or a fragmentedglycopeptide.

A. Stationary Phase Optimization (FIGS. 1-2 and FIG. 5)

LC Condition

-   -   Instrument: ACQUITY UPLC™ H-Class Bio (Waters Corporation,        Milford, Mass.)    -   Column: narrow bore column (2.1×150 mm) with various HILIC        sorbents    -   HILIC sorbents:

Core Rho N Prototype d_(p) (Core d_(p)/ APD N Coverage Name Description(μm) Particle d_(p)) (Å) % μmol/m² A 1.7 μm d_(p) Amide-bonded BEH n/an/a ~45 1.7 2.52 B 1.7 μm d_(p) Amide-bonded BEH n/a n/a ~300 1.3 10.19C 1.9 μm d_(p) Amide Bonded Superficially Porous 1.57 0.84 324 0.3 9.47(Rho = 0.84) Silica D 1.7 μm d_(p) Amide Bonded Superficially Porous1.57 0.93 232 0.1 6.43 (Rho = 0.93) Silica E 1.7 μm d_(p) Amide-bondedBEH n/a n/a ~300 0.92 7.25 F 1.7 μm d_(p) Amide-bonded BEH n/a n/a ~3001.26 10.12

-   -   Mobile Phase A: 0.1% TFA, H2O    -   Mobile Phase B: 0.1% TFA, ACN    -   Gradient (FIG. 1):20% to 80% H₂O in 20 min    -   Gradient (FIGS. 2 and 5): 20% to 34% H₂O in 1 min, then 34% to        41%    -   H₂O in 20 min    -   Column Temperature: 30-80° C.    -   Flow rate: 0.2 mL/min

MS Condition

-   -   Instrument: Xevo G2 QToF    -   Source temperature: 150° C.    -   Desolvation temperature: 350° C.    -   Capillary voltage: 3.0 kV    -   Sample cone voltage: 45V

B. Mobile Phase Additive (FIG. 3)

LC Condition

-   -   Instrument: ACQUITY UPLC™ H-Class Bio (Waters Corporation,        Milford, Mass.)    -   Column: narrow bore column (2.1×150 mm)    -   HILIC sorbent: 1.7 μm amide-bonded BEH (300 Å APD)    -   Mobile Phase A: H₂O and mobile phase additive    -   Mobile Phase B: Acetonitrile and mobile phase additive    -   Gradient: 20% to 80% H₂O in 20 min    -   Mobile Phase Additive: 0.5% (v/v) formic acid        -   50 mM ammonium formate (pH 4.4)        -   0.1% (v/v) trifluoroacetic acid (TFA)    -   Column Temperature: 80° C.    -   Flow rate: 0.2 mL/min    -   Injection volume: 0.5 μL for 1 μg of protein

C. Column Pressure (FIGS. 4A-B)

LC Condition

-   -   Instrument: ACQUITY UPLC™ H-Class Bio (Waters Corporation,        Milford, Mass.)    -   Column: narrow bore column (2.1×150 mm)    -   HILIC sorbent: 1.7 m amide-bonded BEH (300 Å APD)    -   Mobile Phase A: 0.1% TFA, H₂O    -   Mobile Phase B: 0.1% TFA, acetonitrile    -   Gradient: 20% to 30% H₂O in 1 min, then 30% to 37% H₂O in 20 min    -   Column Pressure at Time of Analyte Elution: 3200 psi, 4500 psi,        and 7200 psi    -   Column Temperature: 30° C.    -   Flow rate: 0.2 mL/min    -   Injection volume: 1.5 μL with 3 μg of intact trastuzumab        (protein)

D. Column Temperature (FIG. 6)

LC Condition

-   -   Instrument: ACQUITY UPLC™ H-Class Bio (Waters Corporation,        Milford, Mass.)    -   Column: narrow bore column (2.1×150 mm)    -   HILIC sorbent: 1.7 m amide-bonded BEH (300 Å APD)    -   Mobile Phase A: 0.1% TFA, H₂O    -   Mobile Phase B: 0.1% TFA, and acetonitrile    -   Gradient: 20% to 80% H₂O in 20 min    -   Column Temperature: 30° C. −80° C.    -   Flow rate: 0.2 mL/min    -   Injection volume: 0.5 μL for 1 μg of protein

E. Flow Rate (FIG. 7)

LC Condition

-   -   Instrument: ACQUITY UPLC™ H-Class Bio (Waters Corporation,        Milford, Mass.)    -   Column: narrow bore column (2.1×150 mm)    -   HILIC sorbent: 1.7 m amide-bonded BEH (300 Å APD)    -   Mobile Phase A: 0.1% TFA, H₂O    -   Mobile Phase B: 0.1% TFA, and acetonitrile a    -   Gradient: 34% to 41% H₂O over various times (20 min for 0.2        mL/min flow rate)    -   Flow rate: 0.1˜0.4 mL/min    -   Column temperature: 30° C.    -   Injection volume: 0.5 μL for 1 μg of protein

F. Sample Solvent Effect (FIG. 5)

LC Condition

-   -   Instrument: ACQUITY UPLC™ H-Class Bio (Waters Corporation,        Milford, Mass.)

Column: narrow bore column (2.1×150 mm)

HILIC sorbent: 1.7 μm amide-bonded BEH (300 Å APD)

Mobile Phase A: 0.1% TFA, H₂O

Mobile Phase B: 0.1% TFA, and acetonitrile

Gradient: 20% to 30% H₂O in 1 min, then 34% to 41% in 20 min

Flow rate: 0.2 mL/min

Column temperature: 30° C.

Injection volume: 0.5˜10 μL for 1 μg of protein (aqueous diluent)

G. Orthogonality with C4 Reversed Phase Column (FIG. 11)

LC Condition

-   -   Instrument: ACQUITY UPLC™ H-Class Bio (Waters Corporation,        Milford, Mass.)    -   Column: narrow bore column (2.1×150 mm)

BEH C4 (300 Å APD) BEH amide-bonded (300 Å APD) Mobile Phase A: 0.1%TFA, H₂O Mobile Phase A: 0.1% TFA, Mobile Phase B: 0.1% TFA, andH₂OMobile Phase B: 0.1% TFA, acetonitrile Gradient: 5% to acetonitrileGradient: 20% to 33.3% acetonitrile in 1 min, then 34% H₂O in 1 min,then 34% to 33.3% to 40.3% acetonitrile in 20 41% H₂O in 20 min Columnmin Column Temperature: 80° C. Temperature: 30° C.

-   -   Flow rate: 0.2 mL/min    -   Injection volume: 0.5 μL for 1 μg protein

Glycosylation Profiling of a Glycosylated Monoclonal Antibody

A. Protein Samples

Preparation of Reduced Trastuzumab

Trastuzumab was reduced under denaturing conditions. Formulatedtrastuzumab was diluted to 1.5 mg/mL into a solution with a finalcomposition of 6 M GuHCl and 80 mM TCEP then incubated at 37° C. for 1hour. Other protocols for trastuzumab were also evaluated, including oneinvolving the use of RapiGest SF surfactant and another involving 80° C.heat denaturation. For the surfactant-assisted reduction, trastuzumabwas diluted to 2 mg/mL into a solution comprised of 0.05% (w/v) RapiGestSF and 40 mM TCEP then incubated at 37° C. for 1 hour. For the procedureinvolving heat denaturation, formulated trastuzumab was diluted to 1.5mg/mL into a solution containing 80 mM TCEP then heated for 10 min at80° C., followed by 50 min at 37° C. Although all procedures yieldedantibody in reduced form, there was significant advantage to theguanidine procedure as, unlike the other procedures, aqueous injectionsof this preparation onto an amide HILIC column produced no indication ofself-associated heavy chains. FIG. 10 shows comparative views ofchromatograms of reduced trastuzumab sample preparations.

IdeS Digestion of Trastuzumab and Cetuximab

Prior to digestion with IdeS, Cetuximab was digested withcarboxypeptidase B to complete the partial removal of the lysineC-terminal residues that is typical of the antibody (Ayoub et al,Correct primary structure assessment and extensive glyco-profiling ofcetuximab by a combination of intact, middle-up, middle-down andbottom-up ESI and MALDI mass spectrometry techniques (MAbs, 5 (5),699-710, 2013). Formulated cetuximab (Erbitux) was mixed withcarboxypeptidase B (223 u/mg) at a ratio of 100:1 (w/w), diluted into 20mM phosphate (pH 7.1), and incubated at a concentration of 1.8 mg/mL for2 hours at 37° C. The carboxypeptidase B treated cetuximab was thenadded to 100 units of IdeS and incubated for 30 minutes at 37° C.Similarly, formulated trastuzumab (Herceptin) was diluted into 20 mMphosphate (pH 7.1), added to 100 units of IdeS, and incubated at aconcentration of 3 mg/mL for 30 minutes at 37° C.

Both of the resulting IdeS-digested antibodies were denatured andreduced by the addition of 1M TCEP and solid GuHCl. The final buffercomposition for the denaturation/reduction step was approximately 6 MGuHCl, 80 mM TCEP, and 10 mM phosphate (pH 7.1). IdeS-digested cetuximab(0.9 mg/mL) and IdeS-digested trastuzumab (1.5 mg/mL) were incubated inthis buffer at 37° C. for 1 hour, prior to being stored at 4° C.

Digestion of Trastuzumab to Peptides

An adaptation of a previously published procedure was employed toprepare a non-reduced Lys-C digest of trastuzumab (Lauber et al.,High-Resolution Peptide Mapping Separations with MS-Friendly MobilePhases and Charge-Surface-Modified C18. Anal Chem 2013, 85 (14),6936-44.; Richardson et al, Automated in-solution protein digestionusing a commonly available high-performance liquid chromatographyautosampler. Anal Biochem 2011, 411 (2), 284-91.). The antibody wasfirst denatured under non-reducing conditions in the presence ofiodoacetamide, which serves to alkylate free thiols and thereby minimizedisulfide scrambling. The antibody was diluted to 2.5 mg/mL into adenaturing buffer with a final composition of 5.8 M GuHCl, 0.5 mMiodoacetamide, and 0.1 M phosphate (pH 7.1) then incubated for 2 hoursat 37° C. Subsequently, the denatured trastuzumab was diluted to 0.4mg/mL with a urea containing buffer and mixed with Achromobacterprotease I (Lys-C) at a 20:1 w/w ratio. The final buffer compositionduring digestion was 3 M Urea, 1 M GuHCl, 40 mM NH₂OH, 0.08 mMiodoacetamide, and 0.1 M phosphate (pH 7.1). Lys-C digests wereincubated at 37° C. for 16 hours, before being quenched by acidificationwith TFA, and stored at −80° C. until analyzed.

Digestion of Cetuximab to Peptides

Reduced and alkylated cetuximab (past expiry) was digested with acombination of Achromobacter protease I (Lys-C) and trypsin. Formulatedcetuximab was concentrated to 10 mg/mL and buffer exchanged with a 10kDa MWCO centrifugal filter (Millipore, Billerica, Mass.) into asolution of 6 M GuHCl, 50 mM DTT, and 0.2 M phosphate (pH 8.1), thenincubated at 37° C. for 2 hours. Thereafter, the sample was diluted witha solution of iodoacetamide, bringing the antibody concentration to 8mg/mL and the buffer composition to 4.8 M GuHCl, 40 mM DTT, 50 mMiodoacetamide, and 0.17 M phosphate (pH 8.1). Alkylation withiodoacetamide was allowed to proceed under these conditions for 10 minin the dark at 37° C., before being quenched by the addition ofcysteine, diluted with a urea-containing buffer, and mixed withAchromobacter protease I (Lys-C) at a 4:1 w/w ratio. The resultingdigest solution of 0.8 mg/mL cetuximab, 0.5 M GuHCl, 3 M Urea, 40 mMNH₂OH, 4 mM DTT, 5 mM iodoacetamide, 6 mM cysteine, and 0.1 M phosphate(pH ˜7.1) was incubated at 37° C. After 2 hours of incubation, thisdigest solution was diluted two fold with water and an aliquot oftrypsin, such that the protein:trypsin ratio was 4:1 (w/w). Afterincubation at 37° C. for another 2 hours, the digest solution was againdiluted two fold with water and a fresh aliquot of trypsin. With a totalprotein:trypsin ratio of 2:1 (w/w), the digest was left to incubate at37° C. for 16 hours. Following this incubation, the digest was quenchedby acidification with TFA and stored at −80° C. until analyzed.

B. HILIC Chromatography and Mass Spectrometry for Characterization ofIntact, Glycosylated Monoclonal Antibodies and their Fragments

Glycoforms of intact trastuzumab were separated and analyzed using the1.7 m poly-amide bonded, wide-pore BEH (300 Å APD) stationary phase, aWaters ACQUITY UPLC™ H-Class Bio (Waters Corporation, Milford, Mass.)and Xevo G2 QTof mass spectrometer (Waters Corporation, Milford Mass.).An aqueous solution of trastuzumab was injected onto a 2.1 mm ID columnin a volume of 1.5 μL. The injected sample (3 μg) was separated using aflow rate 0.2 mL/min, column temperature of 30° C., and a gradientconsisting of 1 min ramp from 20 to 30% aqueous mobile phase followed bya 20 min ramp from 30 to 37% aqueous mobile phase (aqueous mobile phase:0.1% (v/v) TFA in water; organic mobile phase: 0.1% (v/v) TFA in ACN).FIGS. 4A-B and FIG. 6 show examples of these separations. Methodssimilar to those described were also applied to HILIC based separationsof glycosylated heavy chains derived from trastuzumab as well asglycosylated fragments originating from trastuzumab IdeS digests. Inthese analyses, 1 μg of sample was injected in 0.67 μL from aqueousdiluent containing high concentrations of denaturant (6 M GuHCl). Thesame methods were applied to separate the glycosylated species presentin an IdeS digest of carboxypeptidase B treated cetuximab, though inthis separation, a column temperature of 60° C. was used to achievedesired selectivity among glycoforms. FIGS. 8A-B, 11, 14, and 15 showexamples of these separations.

Reversed phase (RP) separations of IdeS-fragmented trastuzumab wereperformed to demonstrate the orthogonality between RP and the describedHILIC separations. A BEH300 C4 column (2.1×150 mm, 1.7 μm, 300 Å,Waters, Milford, Mass.) was used to chromatograph the samples at a flowrate of 0.2 mL/min and temperature of 80° C. across a linear gradientconsisting of a 1 min ramp from 5 to 33.3% organic mobile phase followedby a 20 min ramp from 33.3 to 40.3% organic mobile phase (aqueous mobilephase: 0.1% (v/v) TFA in water; organic mobile phase: 0.1% (v/v) TFA inACN). FIG. 11 presents a comparison of a reversed phase C4 versus HILICamide-bonded BEH (300 Å APD) based separation of IdeS-fragmentedtrastuzumab.

Species eluting during the above separations were detected serially viaUV absorbance at 214 and 280 nm (2 Hz scan rate) followed by onlineESI-MS. Mass spectra were acquired with a Xevo G2 QToF operating with acapillary voltage of 3.0 kV, source temperature of 150° C., desolvationtemperature of 350° C., and sample cone voltage of 45 V. Mass spectrawere acquired at a rate of 2 Hz with a resolution of approximately20,000 over a range of 500-4000 m/z.

Deconvolution of Mass Spectra

Raw ESI mass spectra were deconvoluted using MassLynx (V4.1) andMaxEnt 1. Deconvolution parameters were as follows: m/z input range of800-3200 (RNase B), 800-3800 (IdeS mAb fragments), and 2000-4000 (intactmAb); output mass range set to 10,000-20,000 (RNase B), 20,000-40,000(IdeS mAb fragments), and 140,000-160,000 (intact mAb); outputresolution set to 0.1 Da/channel (RNase B), 0.5 Da/channel (IdeS mAbfragments), and 1 Da/channel (intact mAb); width at half-height foruniform Gaussian model set to 1 Da (RNase B and IdeS mAb fragments) and1.5 Da (intact mAb); minimum intensity ratio left and right set to 33%;maximum number of iterations defined as 15.

Profiling Lot-to-Lot Variation of Glycosylation

Different batches of trastuzumab were characterized in duplicate at theIdeS fragment level of analysis as described above as a means to studylot-to-lot variability of the glycan profiles of a mAb. UV chromatogramswere integrated using UNIFI V1.6 and major components/segments werequantified in the form of relative abundance. FIG. 14 shows a set ofchromatograms corresponding to IdeS-digested trastuzumab from differentmanufactured batches as obtained with the stationary phase material inthe invention. Integration and relative quantitation of the different,resolved glycoforms is demonstrated.

C. HILIC Chromatography and Mass Spectrometry for Glycopeptide Mapping

Tryptic and Lys-C digests of the monoclonal antibodies (trastuzumab andcetuximab) were analyzed by HILIC-UV-MS with a Waters ACQUITY UPLC™H-Class Bio (Waters Corporation, Milford, Mass.) and Synapt G2-S massspectrometer (Waters Corporation, Milford, Mass.).

In preparation for HILIC chromatography, aqueous Lys-C and trypticdigests were diluted in a ratio of 1:4 with acetonitrile and centrifugedat 16×1000 g for 10 minutes to remove any insoluble composition.Supernatant from the centrifuged digest was thereafter injected.

Results typical of these analyses are shown in FIGS. 9 and 16. FIG. 9 isa chromatogram of Lys-C-digested trastuzumab characterized with thestationary phase material in the invention. FIG. 16 presents achromatogram corresponding to tryptic glycopeptide mapping oftrastuzumab obtained with a hydrophilic, poly-amide bonded stationaryphase with 300 Å pores (the stationary phase material in the invention).

Stationary Phase Optimization (FIG. 12)

LC Condition

-   -   Instrument: Waters ACQUITY UPLC™ H-Class Bio (Waters        Corporation, Milford, Mass.)    -   Column: narrow bore column (2.1×150 mm) with various HILIC        sorbents    -   HILIC sorbents: 1.7 μm amide-bonded BEH (130 Å APD)        -   1.7 μm amide-bonded BEH (300 Å APD)    -   Mobile Phase A: 0.1% TFA, H2O    -   Mobile Phase B: 0.1% TFA, ACN    -   Gradient: 20% to 50% H2O in 60 min    -   Column Temperature: 30-60° C.    -   Flow rate: 0.2 mL/min    -   Detection: TUV detector (500 nL flow cell, 10 Hz scan rate),        Absorbance at 214 nm

For the first 5 min of each run, effluent was diverted to waste, ratherthan the ESI source.

MS Condition

-   -   Instrument: Waters Synapt G2-S mass spectrometer Source        temperature: 120° C.    -   Desolvation temperate: 350° C.    -   Capillary voltage: 3.0 kV    -   Sample cone voltage: 25 V    -   Mass spectra were acquired at a rate of 10 Hz with a resolution        of approximately 20,000 over a range of 50-2500 m/z.

In addition, real-time mass correction was employed via lockspray with asolution of 0.1 μM Glu-fibrinopeptide B in 50:50 water/ACN, 0.1% (v/v)FA. This calibrant was infused at a flow rate of 20 l/min and sampledevery 1 min.

Identification of Glycosylation of Monoclonal Antibody

Data acquired during these separations were analyzed through automatedinterpretation with BiopharmaLynx (V 1.3.3) as well as manualinterpretation with MassLynx (V4.1). Glycopeptide identifications weremade through matching experimental masses to the theoretical masses ofpeptides generated by in silico protein digestion and consideration ofglycans previously reported for trastuzumab and cetuximab (MAbs 2013, 5(5), 699-710; Methods Mol Biol 2013, 988, 93-113; Anal Biochem 2007, 364(1), 8-18). Glycopeptides from trastuzumab were identified down to a 1%ion intensity level relative to the most abundant glycopeptide, and thecomposition of each unique chromatographic peak was interrogated to a 5%ion intensity level relative to the most abundant glycopeptide detectedat that retention time. Glycopeptides from cetuximab were similarlyidentified at relative intensities of 2% and 10%, respectively. Massesobserved for identified glycopeptides were found to be in agreement withtheoretical masses to within 11 ppm.

Released Glycan Analysis

2-AB labeled, released N-linked glycans from trastuzumab were preparedand analyzed alongside the glycopeptide mapping analysis of trastuzumab,in order to benchmark a current state-of-the-art approach for glycananalysis (Houel, S.; Yu, Y. Q.; Cosgrave, E.; Chen, W., Comparison ofReleased N-Glycans between the Innovator and a Candidate Biosimilar: ACase Study on Etanercept In HPLC, Amsterdam, 2013). N-linked glycanswere released and labeled with 2-AB using a GlycoWorks High-ThroughputSample Preparation Kit (176003090, Waters, Milford, Mass.). Formulatedtrastuzumab was diluted to 0.5 mg/mL into a buffer containing 0.1% (w/v)RapiGest SF surfactant, 5 mM DTT, and 25 mM ammonium bicarbonate (pH7.8) then incubated for 30 min at 37° C. This was followed with analkylation step, wherein the mAb was subjected to a 30 min roomtemperature incubation with 10 mM iodoacetamide. The mAb, still at anapproximate concentration of 0.5 mg/mL, was thereafter subjected toPNGase F (Glyko N-Glycanase, GKE 5006A, Prozyme, Hayward, Calif.) in a25 mM ammonium bicarbonate (pH 7.8) buffer containing approximately 0.1%(w/v) RapiGest SF for 16 hours at a 5:1 ratio of enzyme activityconcentration (U/mL) to protein weight concentration (mg/mL). Releasedglycans were thereafter extracted from the PNGase F digest using aGlycoWorks HILIC telution plate (186002780, Waters, Milford, Mass.). The5 mg of HILIC solid phase extraction (SPE) sorbent (packed in anindividual well of the telution plate) was conditioned with 200 μLvolumes of water followed by 200 μL of 15:85 water/ACN. Subsequently, 50μL of the PNGase F digest was diluted to 400 μL with ACN and loaded ontothe SPE sorbent. The adsorbed sample was then washed 3 times with 200 μLof 85% ACN. After this, glycans were eluted with 3, 50 μL volumes of 100mM ammonium acetate (pH 7), 5% ACN, and the obtained eluate was driedunder vacuum. To convert glycosylamine terminated glycans to glycanswith free reducing termini, dried eluate was reconstituted in 50 μL of1% (v/v) formic acid, 50:50 water/ACN, incubated at room temperature for40 min, and thereafter dried under vacuum.

Following acid-treatment, the glycans were labeled with 2-AB by means ofa reductive amination reaction: dried glycans were reconstituted in a 10μL volume of 30:70 acetic acid/DMSO containing 90 mM 2-AB and 240 mMsodium cyanoborohydride prior to being incubated at 65° C. for 3 hours.The resulting 2-AB labeled glycans were then extracted from the 2-ABlabeling mixture using HILIC SPE just as the unlabeled, released glycanshad been extracted from the PNGase F digest. Eluate obtained from thisfinal SPE clean-up was dried under vacuum, reconstituted in 40:60water/ACN, and stored at 4° C. until analyzed.

Separations and analyses of 2-AB labeled N-glycans were performed byHILIC-FLR-MS with a Waters ACQUITY UPLC™ H-Class Bio (WatersCorporation, Milford, Mass.), ACQUITY FLR detector (Waters Corporation,Milford, Mass.), and Synapt G2-S mass spectrometer (Waters Corporation,Milford, Mass.). Labeled N-glycans prepared from approximately 9 μg oftrastuzumab were loaded and separated on a narrow-bore column packedwith poly-amide bonded, standard pore diameter BEH particles (2.1×150mm, 1.7 m, 130 Å) using conditions that have previously been employed toassign glucose unit (GU) values to chromatographic peaks and tentativelyidentify species using GlycoBase (Houel et al., Comparison of ReleasedN-Glycans between the Innovator and a Candidate Biosimilar: A Case Studyon Etanercept In HPLC, Amsterdam, 2013; Campbell et al., GlycoBase andautoGU: tools for HPLC-based glycan analysis. Bioinformatics, 24 (9),1214-1216, 2008) Briefly, separations were conducted with a columntemperature of 40° C., a flow rate of 0.4 mL/min, and a methodconsisting of a 2.06 min hold at 30% aqueous mobile phase followed by abinary, linear gradient from 30 to 47% aqueous mobile phase over 32.74min (aqueous mobile phase: 50 mM ammonium formate, pH 4.4; organicmobile phase: ACN). Eluting species were serially detected byfluorescence (2 μL flow cell; excitation/emission at 360/428 nm; 10 Hzscan rate, Gain=1) and ESI-MS. The Synapt G2-S was operated with thefollow settings: capillary voltage of 3.0 kV, source temperature of 120°C., desolvation temperature of 350° C. and sample cone voltage of 80 V.Mass spectra were acquired at a rate of 2 Hz with a resolution ofapproximately 20,000 over a range of 500-2500 m/z. Mass correction wasemployed real-time via lockspray with a solution of 0.1 μMGlu-fibrinopeptide B in 50:50 water/ACN, 0.1% (v/v) formic acid infusedat a flow rate of 20 μl/min and sampled every 1 min.

FIG. 13 presents chromatograms corresponding to Lys-C glycopeptidemapping of trastuzumab obtained with a hydrophilic, poly-amide bondedstationary phase with 300 Å pores versus analysis of anthranilamidelabeled, released N glycans (from trastuzumab) obtained with ahydrophilic, poly-amide bonded stationary phase with 130 Å pores.

Protein Sequence Data

The sequences of trastuzumab and cetuximab were acquired from previouspublication (Harris et el., Identification of multiple sources of chargeheterogeneity in a recombinant antibody. J Chromatogr B Biomed Sci Appl,752 (2), 233-45, 2001) and IMGT/mAb-DB (Poiron et. al, IMGT/mAb-DB: theIMGT® database for therapeutic monoclonal antibodies. In JOBIM,Montpellier, France, 2010).

INCORPORATION BY REFERENCE

The entire contents of all patents, published patent applications andother references cited herein are hereby expressly incorporated hereinin their entireties by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents were consideredto be within the scope of this invention and are covered by thefollowing claims.

1-99. (canceled)
 100. A porous material comprising a copolymer comprising at least one hydrophilic monomer and a first poly-amide bonded phase and a second poly-amide bonded phase, wherein: the average pore diameter is greater than or equal to about 200 Å; and the porous material comprises: a porous or nonporous organic-inorganic hybrid core comprising an aliphatic bridged silane, at least one hydrophilic monomer grafted to the core, and the first and the second poly-amide bonded phases are bonded to the at least one hydrophilic monomer.
 101. The porous material of claim 100, wherein the porous material comprises a porous particle that comprises said copolymer.
 102. The porous material of claim 100, wherein the porous material comprises a porous monolith that comprises said copolymer.
 103. The porous material of claim 100, wherein the first poly-amide bonded phase is derived from acrylamide, divinylbenzene, styrene, ethylene glycol dimethacrylate, 1-vinyl-2-pyrrolidinone and tert-butylmethacrylate, methacrylamide, N,N′-(1,2-dihydroxyethylene)bisacrylamide, N,N′-ethylenebisacrylamide, N,N′-methylenebisacrylamide, butyl acrylate, ethyl acrylate, methyl acrylate, 2-(acryloxy)-2-hydroxypropyl methacrylate, N,N-bis(2-cyanoethyl)acrylamide, N-acryloyltris(hydroxymethyl)aminomethane, 3-(acryloxy)-2-hydroxypropyl methacrylate, trimethylolpropane triacrylate, trimethylolpropane ethoxylate triacrylate, tris[(2-acryloyloxy)ethyl]isocyanurate, acrylonitrile, methacrylonitrile, itaconic acid, methacrylic acid, trimethylsilylmethacrylate, N-[tris(hydroxymethyl)methyl]acrylamide, (3-acrylamidopropyl)trimethylammonium chloride, [3-(methacryloylamino)propyl]dimethyl(3-sulfopropyl)ammonium hydroxide inner salt,


104. The porous material of claim 100, wherein the first poly-amide bonded phase is derived from acrylamide.
 105. The porous material of claim 100, wherein the second poly-amide bonded phase is derived from N,N-methylenebisacrylamide, N,N-ethylenebisacrylamide, N,N-propylenebisacrylamide, N,N-butylenebisacrylamide, N,N′-(1,2-dihydroxyethylene)bisacrylamide, or 1,4-bis(acryloyl)piperazine.
 106. The porous material of claim 100, wherein the first poly-amide bonded phase is present in 35 to 99 mole % of the poly-amide bonded phases.
 107. The porous material of claim 100, wherein the second poly-amide bonded phase is present in 65 to 1 mole % of the poly-amide bonded phases.
 108. The porous material of claim 100, wherein said hydrophilic monomer is 3-methacryloxypropyltrichlorosilane, 3-methacryloxypropylmethyldichlorosilane, 3-methacryloxypropyldimethylchlorosilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyldimethylmethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyldimethylethoxysilane, 3-acryloxypropyltrichlorosilane, 3-acryloxypropylmethyldichlorosilane, 3-acryloxypropyldimethylchlorosilane 3-acryloxypropyltrimethoxysilane, 3-acryloxypropylmethyldimethoxysilane, 3-acryloxypropyldimethylmethoxysilane, 3-acryloxypropyltriethoxysilane, 3-acryloxypropylmethyldiethoxysilane, 3-acryloxypropyldimethylethoxysilane, styrylethyltrichlorosilane, styrylethylmethyldichlorosilane, styrylethyldimethylchlorosilane, styrylethyltrimethoxysilane, styrylethylmethyldimethoxysilane, styrylethyldimethylmethoxysilane, styrylethyltriethoxysilane, styrylethylmethyldiethoxysilane, styrylethyldimethylethoxysilane, vinyltriethoxysilane, vinyltrimethoxysilane, N-(3-acryloxy-2-hydroxypropyl)-3-aminopropyl triethoxysilane, (3-acryloxypropyl) trimethoxysilane, O-(methacryloxyethyl)-N-(triethoxysilylpropyl)urethane, N-(3-methacryloxy-2-hydroxypropyl)-3-aminopropyl triethoxysilane, methacryloxy methyltriethoxysilane, methacryloxymethyl trimethoxysilane, methacryloxypropy methyldiethoxysilane, methacryloxypropyl methyldimethoxysilane, methacryl oxypropyltris (methoxyethoxy)silane, 3-(N-styrylmethyl-2-aminoethylamino) propyltrimethoxysilane hydrochloride,


109. The porous material of claim 100, wherein the porous material has a median pore diameter of about 100 Å to about 1000 Å.
 110. The porous material of claim 100 wherein the porous material has a nitrogen content of said material is from about 0.5% N to about 20% N as measured by combustion analysis.
 111. A method for removing or isolating a component from a mixture comprising: contacting the mixture with a chromatographic material comprising a porous material to thereby remove or isolate the component from the mixture, the porous material comprising a copolymer comprising at least one hydrophilic monomer and a first poly-amide bonded phase and a second poly-amide bonded phase, wherein: the average pore diameter is greater than or equal to about 200 Å; and the porous material comprises: a porous or nonporous organic-inorganic hybrid core comprising an aliphatic bridged silane, at least one hydrophilic monomer grafted to the core, and the first and the second poly-amide bonded phases are bonded to the at least one hydrophilic monomer.
 112. The method of claim 111, wherein the porous material comprises a porous particle that comprises said copolymer.
 113. The method of claim 111, wherein the porous material comprises a porous monolith that comprises said copolymer.
 114. The method of claim 111, wherein the first poly-amide bonded phase is derived from acrylamide, divinylbenzene, styrene, ethylene glycol dimethacrylate, 1-vinyl-2-pyrrolidinone and tert-butylmethacrylate, methacrylamide, N,N′-(1,2-dihydroxyethylene)bisacrylamide, N,N′-ethylenebisacrylamide, N,N′-methylenebisacrylamide, butyl acrylate, ethyl acrylate, methyl acrylate, 2-(acryloxy)-2-hydroxypropyl methacrylate, N,N-bis(2-cyanoethyl)acrylamide, N-acryloyltris(hydroxymethyl)aminomethane, 3-(acryloxy)-2-hydroxypropyl methacrylate, trimethylolpropane triacrylate, trimethylolpropane ethoxylate triacrylate, tris[(2-acryloyloxy)ethyl] isocyanurate, acrylonitrile, methacrylonitrile, itaconic acid, methacrylic acid, trimethylsilylmethacrylate, N-[tris(hydroxymethyl)methyl]acrylamide, (3-acrylamidopropyl)trimethylammonium chloride, [3-(methacryloylamino)propyl]dimethyl(3-sulfopropyl)ammonium hydroxide inner salt,


115. The method of claim 111, wherein the first poly-amide bonded phase is derived from acrylamide.
 116. The method of claim 111, wherein the second poly-amide bonded phase is derived from N,N-methylenebisacrylamide, N,N-ethylenebisacrylamide, N,N-propylenebisacrylamide, N,N-butylenebisacrylamide, N,N′-(1,2-dihydroxyethylene)bisacrylamide, or 1,4-bis(acryloyl)piperazine.
 117. The method of claim 111, wherein the first poly-amide bonded phase is present in 35 to 99 mole % of the poly-amide bonded phases.
 118. The method of claim 111, wherein the second poly-amide bonded phase is present in 65 to 1 mole % of the poly-amide bonded phases.
 119. The method of claim 111, wherein said hydrophilic monomer is 3-methacryloxypropyltrichlorosilane, 3-methacryloxypropylmethyldichlorosilane, 3-methacryloxypropyldimethylchlorosilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyldimethylmethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyldimethylethoxysilane, 3-acryloxypropyltrichlorosilane, 3-acryloxypropylmethyldichlorosilane, 3-acryloxypropyldimethylchlorosilane 3-acryloxypropyltrimethoxysilane, 3-acryloxypropylmethyldimethoxysilane, 3-acryloxypropyldimethylmethoxysilane, 3-acryloxypropyltriethoxysilane, 3-acryloxypropylmethyldiethoxysilane, 3-acryloxypropyldimethylethoxysilane, styrylethyltrichlorosilane, styrylethylmethyldichlorosilane, styrylethyldimethylchlorosilane, styrylethyltrimethoxysilane, styrylethylmethyldimethoxysilane, styrylethyldimethylmethoxysilane, styrylethyltriethoxysilane, styrylethylmethyldiethoxysilane, styrylethyldimethylethoxysilane, vinyltriethoxysilane, vinyltrimethoxysilane, N-(3-acryloxy-2-hydroxypropyl)-3-aminopropyl triethoxysilane, (3-acryloxypropyl) trimethoxysilane, O-(methacryloxyethyl)-N-(triethoxysilylpropyl)urethane, N-(3-methacryloxy-2-hydroxypropyl)-3-aminopropyl triethoxysilane, methacryloxy methyltriethoxysilane, methacryloxymethyl trimethoxysilane, methacryloxypropy methyldiethoxysilane, methacryloxypropyl methyldimethoxysilane, methacryl oxypropyltris (methoxyethoxy)silane, 3-(N-styrylmethyl-2-aminoethylamino) propyltrimethoxysilane hydrochloride,


120. The method of claim 111, wherein the porous material has a median pore diameter of about 100 Å to about 1000 Å.
 121. The method of claim 111 wherein the porous material has a nitrogen content of said material is from about 0.5% N to about 20% N as measured by combustion analysis. 